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Received 3 February 1998; accepted after revision 27 April 1998.
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ABSTRACT |
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INTRODUCTION |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Endothelin-1 (ET), a potent vasoactive peptide originally discovered in the supernatant of porcine endothelial cells, is released from airway epithelial cells and exerts an important autocrine/paracrine function in the lung (reviewed by Filep, 1992; Markewitz et al. 1995; Michael & Markewitz, 1996). Beside a possible role in the regulation of airway muscle tone and proliferation, ET may trigger cytokine release and modulate immune responses. In the respiratory epithelium, ET was found to increase transepithelial Cl- transport, to stimulate surfactant secretion, to augment mucociliary clearance and to enhance epithelial cell growth.
Ca2+ plays a fundamental role in the various actions of ET. ET usually causes a biphasic Ca2+ signal, consisting of a transient [Ca2+]i increase followed by a lesser but sustained increment lasting for several minutes (reviewed in Highsmith et al. 1992; Pollok et al. 1995; Stojilkovic & Catt, 1996; Schramek & Dunn, 1997). It is generally assumed that this biphasic signal results from both intracellular Ca2+ release and Ca2+ entry across the plasma membrane. The mechanisms by which ET elevates Ca2+ entry are still a matter of controversy and appear to exhibit considerable heterogeneity between different cell types: a role for L-type voltage-dependent calcium channels (L-VDCCs) has been demonstrated in several tissues, but stimulatory as well as inhibitory effects of ET on L-VDCCs have been reported. The most direct evidence in support of a stimulation of L-VDCCs by ET was derived from patch clamp studies in smooth muscle cells (Goto et al. 1989) and ventricular myocytes (Lauer et al. 1992). On the other hand, inhibition of L-VDCCs was observed in the heart (Ono et al. 1994; Xie et al. 1996), in smooth muscle cells (Van Renterghem et al. 1988; Klöckner & Isenberg, 1991; Ohshima et al. 1994) and in pituitary lactotrophs (Lachowicz et al. 1997).
Besides L-VDCCs, Ca2+ entry through non-voltage-gated pathways appears to be a common mechanism in various cell types studied so far (Highsmith et al. 1992; Pollock et al. 1995; Schramek & Dunn, 1997). Consistently, ET may induce Ca2+ entry in cells where KCl depolarization has no effect (Gardner et al. 1992). For this type of Ca2+ entry, non-selective cation channels are potential candidates. In smooth muscle cells and fibroblasts, ET was found to stimulate a non-selective cation current (Van Renterghem et al. 1988; Chen & Wagoner, 1991; Inazu et al. 1994; Enoki et al. 1995; Nakajima et al. 1996). The most detailed description of this was presented by Enoki et al. (1995), who calculated a permeability ratio for Ca2+ over Cs+ of 2·5. Since this current was expressed in cells transfected with cDNA for recombinant ET receptors of the ETA subtype, it most probably proceeds through a ligand-gated ion channel. Finally, store-operated Ca2+ channels were reported to be involved in ET-induced Ca2+ entry (Kruger et al. 1995).
The physiological role of Cl- channels during stimulation with ET is not clear but it is believed to constitute an intermediate step within a cascade of reactions finally leading to the activation of L-VDCCs (Klöckner & Isenberg, 1991; Van Renterghem & Lazdunski, 1993; Salter & Kozlowski, 1996). Most ET-induced Cl- currents studied so far have been described as Ca2+ dependent, and their transient or oscillatory activation is considered to reflect inositol 1,4,5-trisphosphate-induced changes in [Ca2+]i. Because [Cl-] is above the electrochemical equilibrium in most cells, the depolarizing action of this current is expected to activate L-VDCCs. To our knowledge, two types of Ca2+-independent Cl- channel activated in response to ET have been described: a maxi Cl- channel in gastric cells (Kajita et al. 1995) and a very low conductance Cl- channel in smooth muscle cells (Van Renterghem & Lazdunski, 1993). Neither of these channels has been assigned a clear physiological function.
In this report we extend previous concepts about ET-induced ion channels by demonstrating a distinct Cl- current which appears to control, rather than be controlled by, DHP-insensitive Ca2+ entry. Consequently, pharmacological modulation of Cl- channels may offer a potential new approach for controlling the biological actions of ET.
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METHODS |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Cell culture and microelectrode experiments
The procedures have recently been described in detail (Dietl et al. 1995). In short, L2 cells (an epithelial cell line from the rate lung; cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10 % fetal calf serum, 100 U ml-1 penicillin, 100 µg ml-1 streptomycin and 44 mM NaHCO3, between their 24th and 60th passage) were seeded on glass coverslips 1-3 days prior to the experiment. For the experiment, a glass coverslip was mounted into a perfusion chamber allowing rapid exchange of bath solutions. The control bath solution contained (mM): 135 NaCl, 5 KCl, 1 CaCl2, 1 MgCl2, 5 glucose, 10 Hepes (pH 7·4). In low-Cl- solutions, NaCl and KCl were replaced by sodium gluconate and potassium gluconate, respectively. For the measurement of membrane potential (Vm), cells at a subconfluent state were studied under a Zeiss IM3 microscope. Cells were impaled with microelectrodes (made from microfilamented borosilicate tubes, resistance 100-200 M
, tip potential < 5 mV), filled with 1 M KCl. Tip resistance was controlled during each experiment (experiments with changing tip resistances were discarded) by short (0·5 s) repetitive current pulses, resulting in small upward deflections of the Vm traces. The microelectrodes were connected to a self-made high-input impedance electrometer, and Vm was recorded on a strip chart recorder. Experimental solutions were applied after a stable recording of Vm more negative than -40 mV had been obtained.
Perforated cell patch clamp experiments
Perforated patch clamp measurements of the whole-cell current were made as recently described (Schobersberger et al. 1997) with the following modifications. The control bath solution contained (mM): 140 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 5 glucose, 10 Hepes (pH 7·4). The control pipette solution contained (mM): 138 potassium gluconate, 1 MgCl2, 0·1 EGTA, 10 Hepes (pH 7·3), and 300 µg ml-1 amphotericin B (freshly prepared every day from a stock solution of 60 mg ml-1 in dimethyl sulphoxide). Current-voltage relationships were determined by voltage ramps of 3 s duration from a holding potential of -65 to +65 mV, using a pCLAMP 5.5 and 6.1 (Axon Instruments) routine. The currents were measured with an EPC7 amplifier and stored at a sampling rate of 2 kHz on a 486 computer.
Fura-2 experiments
The measurement of [Ca2+]i was performed as described by Delles et al. (1995). In short, coverslips with L2 cells were exposed to 2 µM fura-2 AM for 15 min and then mounted into a perfusion chamber allowing rapid fluid exchange. The cells were exposed to the control solution (see above) for 10 min before experimental solutions were applied. In the low-Cl- solution, added Ca2+ was raised to 3 mM in order to compensate for chelation of Ca2+ with gluconate-, as described by Hosoki & Iijima (1994). Measurements were made in individual cells under an inverted microscope. Monochromatic light was provided at 340 and 380 nm wavelengths alternately for 20 ms (followed by 940 ms of darkness) by an Iobin & Yvon diffraction grating mounted on a high speed scanner. Excitation light was directed through a quartz glass fibre to a grey filter (transmission 3 %) before entering the microscope. The emitted fluorescence was directed through a 420 nm cut-off filter to a photomultiplier tube. To collect fluorescence from a single cell, a 640 µm pin hole was placed in the image plane of the phototube. For the calculation of [Ca2+]i, the minimal fluorescence ratio (Rmin) was determined by addition of ionomycin (20 µM) to a Ca2+-free (5 mM EGTA) bath solution and the maximal fluorescence ratio (Rmax) by addition of ionomycin to the control solution in each cell. Background fluorescence was obtained by quenching fura-2 fluorescence with Mn2+ (5 mM) in the presence of ionomycin at the end of each experiment.
Chemicals
ET was obtained from Calbiochem and fura-2 AM from Molecular Probes. All other chemicals were purchased from Sigma. (+)-Isradipine was kindly provided by Dr Jörg Striessnig (Innsbruck, Austria).
Data analysis and statistics
Digitally stored data were analysed using Datgraf (Cyclobios, Innsbruck, Austria). Data are reported as arithmetic means ±
Electrophysiological effects of ET
Conventional microelectrode measurements represent the 'least-invasive' electrophysiological method of cell recording, because cytosolic ion concentrations are not 'clamped' by an artificial pipette solution. As shown in Fig. 1A, addition of 10 nM ET to the control bath solution evoked a rapid and biphasic depolarization of the membrane potential from -53·8 ± 1·6 to -29·1 ± 0·7 mV (n = 12). The transient hyperpolarizing 'hump' following the initial phase of depolarization (Fig. 1A) was prevented by addition of 5 mM Ba2+ to the bath (n = 6, not shown) and coincided with intracellular Ca2+ release (see below), suggesting that it resulted from a transient activation of Ca2+-dependent K+ channels. The depolarization by ET could be elicited only once, consistent with endothelin-induced retrieval of cell surface ET receptors (Chun et al. 1995) and rapid downregulation of Ca2+ signals (Oles et al. 1997). Likewise, the effect of ET was transient, even in the continuous presence of ET in most cells (Figs 1A and 3A), but still persisted for several minutes (in some cells, full repolarization was not achieved even after 30 min). Since this process fairly exceeds the time course of Ca2+ release or voltage-gated currents (see below), we define it as 'long-lasting', in analogy to another 'long-lasting' ET-induced current described by Enoki et al. (1995).
A, two representative original recordings (analog voltage signals recorded on a strip chart recorder) of the membrane potential (Vm) of two single L2 cells, measured with conventional microelectrodes. The period of cell superfusion with 10 nM endothelin-1 (ET) is indicated above the tracings. Small upward deflections of the voltage traces are due to current injections made in order to control the tip resistance of the microelectrodes. Low Cl- (right-hand trace) indicates replacement of NaCl and KCl by sodium gluconate and potassium gluconate, respectively. B, plot of Vm before (
Perforated patch clamp experiments revealed that this depolarization was caused by the activation of IET, defined as the difference between the maximum current after ET application and the control current (both shown in Fig. 2A) with a Vrev = -22·7 ± 1·8 mV (n = 18). IET exhibited an outward rectification in a control bath solution and averaged -420 ± 16 pA (n = 18) at a pipette potential of -65 mV (I-65 mV) and 1046 ± 28 pA (n = 15) at a pipette potential of 65 mV (I65 mV). This corresponds to an ET-induced increase in cord conductance (i.e. [I65 mV - I-65 mV]/130 mV) of 11·3 nS.
A, representative current-voltage (I-V) relationships of two L2 cells under control conditions (left) and in the presence of low Cl- (right; see Fig. 1A). The I-V relationships in each cell were derived from repetitive voltage ramps in perforated patch clamp experiments (see Methods) before and after the addition of 10 nM endothelin-1 (-ET and +ET, respectively). B, ET-induced currents (IET), defined as the maximum currents after addition of 10 nM ET minus the currents before addition of ET, at a pipette potential of +65 mV (
Effect of extracellular Cl- reduction on ET-induced membrane voltage and current
When bath Cl- was reduced to 6 mM, the ET-induced depolarization was greatly enhanced (Fig. 1A), resulting in a peak Vm of +4·7 ± 2·1 mV (n = 14). This strong depolarization could, in theory, be caused by the activation of a Cl- conductance in the presence of a 'reversed' Cl- concentration gradient (driving Cl- from intracellular to extracellular) and/or by activation of a cation conductance which does not significantly discriminate between Na+ and K+. Nevertheless, the activation of a Cl- conductance is definitely demonstrated by the fact that the change in Vm was negligible when bath Cl- was reduced before ET application, but significant when bath Cl- was replenished during ET application (as reflected by the fast hyperpolarization by 23 ± 4 mV, n = 9; Fig. 1A).
In perforated patch clamp experiments, IET under low-Cl- conditions exhibited a greatly reduced amplitude (Fig. 2A), an altered Vrev of -5·3 ± 6·5 mV (n = 12) and a loss of outward rectification. Accordingly, the conductance increase induced by ET averaged only 1·7 nS, which is only 15 % of that measured with high Cl-. These observations are consistent with microelectrode measurements and reflect the Cl- concentration dependence of the Cl- conductance, yet do not exclude the possibility of simultaneous activation of a non-selective cation conductance. The following experiments were performed to characterize IET further and demonstrate that cations do not significantly contribute to IET.
Effect of cations on ET-induced membrane voltage and current
When bath Na+ was replaced by NMDG+, the ET-induced depolarization was not significantly affected (Fig. 1B). Similarly, removal of bath Ca2+ was without effect (Fig. 1B). These data fit well with perforated patch clamp experiments, indicating no significant reductions of IET by cation replacement: in experiments, where Ca2+ was omitted and Na+ replaced by NMDG+, the I-V relationship of IET was essentially unaltered (Fig. 2B). Accordingly, the Vrev (-27·1 ± 3·3 mV, n = 6) of IET was not significantly different from control.
Effect of replacement of pipette K+ by Cs+ on IET. This was also without effect on IET. Even at +65 mV, i.e. the highest electrical driving force for K+ exit in our experiments, IET was unchanged by intracellular K+ replacement (Fig. 2B).
Effect of Ni2+ on IET. Ni2+, which is commonly used as a blocker of various different cation channels, did not reduce IET at a concentration of 1 mM (Fig. 2B).
The relationship between [Ca2+]i and IET
ET elicited a biphasic Ca2+ signal. An initial transient rise in [Ca2+]i (Figs 3A, 5A and 6) was independent of bath Ca2+ (not shown) and thus a result of intracellular Ca2+ release. A subsequent 'plateau' elevation of [Ca2+]i with variable magnitude was seen in the presence of bath Ca2+ only and is therefore related to Ca2+ entry from the bath into the cytosol. As shown in Fig. 5A, this [Ca2+]i 'plateau' was not always stable but fluctuated, particularly during prolonged exposures to ET. We performed combined perforated patch clamp and fura-2 experiments in single L2 cells in order to disclose a possible direct Ca2+ dependence of IET. An example of such an experiment is shown in Fig. 3A. It is evident that although the change in IET (measured at V = -65 mV) coincided with the rise in [Ca2+]i, IET significantly outlasted the Ca2+ signal, yielding a hysteresis correlation between [Ca2+]i and IET (inset of Fig. 3A). These data indicate that although a trigger function for the generation of IET by Ca2+ is likely, IET is definitely not a Ca2+-dependent current in the general sense.
A, representative original recordings of the whole-cell current at a pipette potential of -65 mV (I-65 mV) and the cytoplasmic Ca2+ concentration ([Ca2+]i), measured in a combined fura-2 and perforated patch clamp experiment in a single L2 cell (see Methods). Same ordinate for I-65 mV (continuous line, negative values) and [Ca2+]i (dotted line, positive values). The addition of 10 nM endothelin-1 (ET) to the non-perfused bath is indicated with an arrow. Currents at other pipette potentials, which were continuously recorded during this experiment, are not shown for simplicity. Inset in A shows the relation between [Ca2+]i and the amount of I-65 mV (-I-65 mV) from the same experiment. Arrows indicate direction of hysteresis as a function of time. B, the effects of thapsigargin. Upper graph: superimposed representative recordings of I-65 mV from two L2 cells, one being treated for 5 min with 200 nM thapsigargin (+ Thapsigargin) before addition of ET (arrow), one being untreated (- Thapsigargin). Middle graph: IET (calculated as in Fig. 2B, at a pipette potential of -65 mV) of cells treated as in the upper graph. Each column represents the mean ±
To elucidate further the Ca2+ dependence of IET, intracellular Ca2+ stores were depleted prior to ET application by a 5 min pretreatment with 200 nM thapsigargin, a selective inhibitor of endoplasmic Ca2+-ATPase. Under these conditions, basal [Ca2+]i was elevated above 200 nM (Fig. 3B, lower graph), presumably through store-operated Ca2+ entry. Furthermore, the transient rise in [Ca2+]i induced by ET was entirely abolished (Fig. 3B, lower graph). On the contrary, ET elicited a significant decline in [Ca2+]i (Fig. 3B, lower graph). Despite this 'inverse' Ca2+ signal, IET could still be activated (Fig. 3B, upper and middle graphs), but the time course of IET activation was significantly slowed down (Fig. 3B, upper graph), and the magnitude of IET was also significantly reduced (Fig. 3B, middle graph). These experiments demonstrate that the elevation of [Ca2+]i is not a prerequisite for the generation of IET.
Effect of (+)-isradipine on IET and [Ca2+]i; effect of ET on L-type Sr2+ currents
DHP-sensitive L-VDCCs play a role in a variety of cellular ET actions. Since L2 cells express these channels (Dietl et al. 1995; Schobersberger et al. 1997), we tested the sensitivity of IET to (+)-isradipine, a potent and highly selective blocker of L-VDCCs. Under control conditions (i.e. in the absence of ET), (+)-isradipine (100 nM) did not affect the magnitude or shape of the I-V relationship elicited by our voltage ramp protocol (n = 6, not shown). As previously shown, however, the L-type Ca2+ (Sr2+) current is completely abolished at the same concentration of (+)-isradipine in L2 cells (Dietl et al. 1995). This indicates that our slow depolarizing voltage ramp (see Methods) failed to elicit transient L-type Ca2+ currents, i.e. our measured net currents are devoid of L-type, voltage-gated current components. Furthermore, (+)-isradipine (100 nM) did not reduce the magnitude of IET (the small reduction in Fig. 2B is not statistically significant), and did not affect the shape of the I-V curve or alter the activation time of IET (not shown). This indicates that the L-type Ca2+ current is neither part of IET nor involved in the activation of IET. Consistently, (+)-isradipine had no apparent effect on the ET-induced Ca2+ signal, i.e. (+)-isradipine neither reduced the peak [Ca2+]i (n = 13) nor affected the shape (oscillating plateau) of the Ca2+ signal (not shown).
To find out if ET affects L-VDCCs in L2 cells, we applied ET under conditions known to activate L-VDCCs repetitively. In the microelectrode experiments, Vm was recorded in the presence of 5 mM Sr2+ (zero Ca2+). Under these conditions, spontaneous repetitive depolarizations occurred (Fig. 4), reflecting inward Sr2+ currents (spikes) through L-VDCCs (described in detail by Dietl et al. 1995). Figure 4 reveals that these Sr2+-induced spikes immediately disappeared upon addition of ET, prior to its depolarizing action (n = 6). Hence, ET inhibited L-VDCCs, as described in other cell types (see above).
Original strip chart recording of cell membrane potential (Vm) from a representative microelectrode experiment as in Fig. 1. [Sr2+] = 5 mM. Cells were superfused with Sr2+ or endothelin-1 (ET, 10 nM) as indicated above the tracing.
Effect of MFA on IET and [Ca2+]i
MFA and related analogues of DPC (diphenylamine-2-carboxylic acid) inhibit several classes of Cl- channels (Mochizuki et al. 1994; Reinspecht et al. 1994; Greenwood & Large, 1995; Okada, 1997). We tested the effect of MFA on both [Ca2+]i and IET. Figure 5A reveals that 100 µM MFA caused a suppression of the 'plateau' [Ca2+]i to basal values and an immediate cessation of [Ca2+]i oscillations. Removal of MFA resulted in a high 'overshoot' of [Ca2+]i (Fig. 5A). When 100 µM MFA was given prior to ET, the ET-induced 'peak' [Ca2+]i (i.e. intracellular Ca2+ release) was also reduced, but not abolished (Fig. 5A). The same concentration of MFA caused an immediate (within seconds) block of IET (Fig. 5B). Notably, this block of IET was much faster than the MFA-induced decline in [Ca2+]i. Hence, we can exclude the possibility that the former was a result of the latter. Figure 5C reveals that MFA exerted dual effects on both IET and the ET-induced 'plateau' [Ca2+]i ([Ca2+]iET), with small but significant increases at 10 µM and an almost complete block at 100 µM. These data suggest that the activation of IET and of Ca2+ entry share some common mechanism.
A, two representative fura-2 measurements of the cytoplasmic Ca2+ concentration ([Ca2+]i) in single L2 cells (not voltage clamped). Superfusion of cells with endothelin-1 (ET, 10 nM) or mefenamic acid (MFA, 10 or 100 µM) is indicated above the tracings. B, superimposed continuous recordings of IET (continuous line, calculated as in Fig. 2A, at a pipette potential of -65 mV) and of the ET-induced 'plateau' cytoplasmic Ca2+ concentration ([Ca2+]iET, dotted line, defined as [Ca2+]i after ET minus [Ca2+]i before ET, measured as in A) in response to 100 µM MFA in two single L2 cells pretreated with 10 nM ET. Same ordinate and time scale for IET and [Ca2+]iET. Note that the inhibition of IET precedes the decline of [Ca2+]iET. C, concentration-response relation of the effects of MFA on [Ca2+]iET (
Effect of low bath Cl- on [Ca2+]i
Owing to this apparent link between IET (which is predominantly a Cl- current) and Ca2+ entry, we tested the effects of ET under low bath Cl- (6 mM) conditions. The results are shown in Fig. 6. It is evident that the entire ET-induced Ca2+ signal was, to a considerable extent, suppressed in the presence of low Cl-. Although we still have no convincing explanation for the smaller peak [Ca2+]i, the reduction of the 'plateau' [Ca2+]i below baseline is consistent with strongly suppressed Ca2+ entry.
The means (continuous lines) and
The results of the present study demonstrate that ET activates a long-lasting current which is primarily carried by Cl- ions and that its suppression - either by the Cl- channel blocker MFA or by removal of bath Cl- - causes severe reduction of the 'plateau' [Ca2+]i, a parameter for DHP-insensitive Ca2+ entry. Notably, IET is not directly activated by Ca2+, although intracellular Ca2+ release enhances the amplitude and accelerates the activation of IET. Since the current is unaffected by Ca2+ removal from the bath, Ca2+ entry does not exert this amplifying function on IET. Taking these findings together, a picture of the ET-induced signalling cascade in L2 cells emerges which modifies current schemes of the interactions between ET-induced currents and Ca2+ signals.
As noted, our data suggest a causal link between IET and Ca2+ entry. One important implication of this would be that Ca2+ entry is controlled by the Cl- current and not vice versa (activation of IET as a result of an elevation of [Ca2+]i can be ruled out by the inverse relation between [Ca2+]i and IET in the presence of thapsigargin; see Fig. 3B). This is supported by the fact that the effect of MFA on IET clearly preceded the change in [Ca2+]i. One drawback of this conclusion could be that MFA exerts independent effects on the ET-stimulated Cl- channel and the Ca2+ entry pathway. In fact, highly selective Cl- channel blockers do not yet exist, and fenamates as well as other Cl- channel blockers have been shown to interfere with several classes of ion channels at the concentrations required to block Cl- currents (Siemer & Gögelein, 1992; Ottila & Toro, 1994; Reinspecht et al. 1994; Enoki et al. 1995; Greenwood & Large, 1995). Although we cannot exclude multiple targets of MFA, the fact that reduction of extracellular Cl- essentially reproduced these effects indicates that a Cl--associated mechanism affects Ca2+ entry by itself and independently of MFA.
Another possibile interpretation of the apparent link between IET and Ca2+ entry would be that the Cl- channel itself is permeable to Ca2+. Apart from theoretical considerations, that a Cl-- and Ca2+-selective but Na+-impermeable ion channel is hardly conceivable, the relation between [Ca2+]i and IET in the presence of thapsigargin (compare with Fig. 3) is not consistent with such a model.
Therefore, the ET-induced Ca2+ entry pathway in L2 cells remains undefined. Although we have recently demonstrated the presence of L-VDCCs in L2 cells (Dietl et al. 1995), we can definitely exclude these channels as being relevant in this context. Two other potential Ca2+ entry pathways, however, should be taken into consideration.
A receptor-operated, Ca2+-permeable, cation channel has recently been described by Enoki et al. (1995). In fact, IET and this cation current share so many properties (non-voltage gated, Ca2+ independent, DHP insensitive, MFA sensitive) that one might speculate that both may be coactivated in L2 cells, but that the cation current is 'hidden' under IET (IET is indeed 10 to 100 times greater than the cation current) and thus 'overlooked' by our experimental protocols. Naturally, we cannot exclude the possibility that we did not detect a small amount of cation current. In other words, it is possible that Ca2+ entry yielded a small current which we were unable to resolve. The major fraction of IET, however, was clearly carried by Cl- and not by Na+, which is evident from our Na+ and Cl- replacement experiments. Hence, in contrast to the finding by Enoki et al. (1995) in smooth muscle cells, we can definitely exclude the possibility that in L2 cells a Ca2+-permeable cation channel is responsible for both the long-lasting depolarization and Ca2+ entry. As discussed above, it is most likely that the Cl- channel, which is responsible for the long-lasting depolarization, and the Ca2+ channel (or Ca2+-permeable cation channel) are two different entities. The most important implication of this is that there is, in contrast to a Ca2+-permeable channel, no a priori correlation between current magnitude and Ca2+ entry.
Since intracellular Ca2+ release is a common effect of ET in various cell types including L2 cells, activation of a store-operated Ca2+ entry pathway is likely to occur and has already been reported to be involved in the action of ET (Kruger et al. 1995). In L2 cells, store-operated Ca2+ entry is suggested by the findings that thapsigargin evoked a long-lasting elevation of [Ca2+]i (see Fig. 3B) and that Ca2+ readdition to Ca2+-depleted L2 cells resulted in a large 'overshoot' of [Ca2+]i (data not shown). An important feature of store-operated Ca2+ entry is its voltage dependence (although it is not voltage gated), because the membrane potential is an important determinant of the electrochemical Ca2+ gradient, driving Ca2+ through very small conductance Ca2+ channels (reviewed by Putney, 1986; Berridge, 1995). This is most directly demonstrated by [Ca2+]i measurements in voltage-clamped cells, where store-operated elevations of [Ca2+]i correlate well with the holding potential through the patch pipette (Leipziger et al. 1994). Consistent with this voltage dependence of store-operated Ca2+ entry is the finding by Dunican et al. (1996) that ET-induced influx of Ca2+ may be modulated by external K+, yielding a positive correlation between the predicted membrane potential and [Ca2+]i. It is easily conceivable, therefore, that the membrane potential - and thus the electrical driving force for Ca2+ - is at least one link between Cl- current and Ca2+ entry. This hypothesis is in aggreement with earlier reports about Cl--sensitive Ca2+ entry by agonists, where extracellular Cl- reduction prevented the sustained [Ca2+]i response to histamine (Hosoki & Iijima, 1994). It is also supported by a combined patch clamp and indo-1 study using thapsigargin-treated endothelial cells (Klishin et al. 1998), although these authors considered a direct interaction of Cl- ions with store-operated Ca2+ channels - rather than the membrane potential - to be the link between Cl- and [Ca2+]i. Several findings of the present study support the idea that the membrane potential functions as the mediator between Cl- current and Ca2+ entry. First, bath Cl- reduction resulted in both a large depolarization (i.e. low electrical driving force for Ca2+) and suppressed [Ca2+]i. Second, ET reduced [Ca2+]i in cells pretreated with thapsigargin (note that IET could still be activated under these conditions). We postulate, therefore, that ET activates both a Cl- channel and a rheogenic store-operated Ca2+ entry, and that the former regulates the latter via the membrane potential. In this context, the physiological importance of the Cl- current would be to reduce rather than to augment Ca2+ entry, because Cl- currents depolarize rather than hyperpolarize most cells. In fact, plateau [Ca2+]i values in response to ET are frequently lower than with other agonists, and, moreover, ET may inhibit [Ca2+]i responses to other agonists (Lachowicz et al. 1997). ET would thus activate the Cl- channel to exert a negative feedback control on its own Ca2+ signal, and possibly on subsequent Ca2+ signals by other agonists.
Information about the structure and mode of activation of this Cl- current is still lacking. We assume that it is a very small conductance channel, most probably similar or identical to the channel described by Van Renterghem & Lazdunski (1993), because in cell-attached patch clamp experiments we were rarely successfull in detecting channel activity, irrespective of the site of ET application (bath or patch pipette). Hence, this channel is obviously different from cAMP- or swelling-activated Cl- channels. Notably, it is also not a common Ca2+-activated Cl- channel as described in several other cell types, where the Cl- current may be taken as an indirect parameter for changes in [Ca2+]i. L2 cells do not appear to express these Ca2+-dependent Cl- channels, because an elevation of [Ca2+]i by ionomycin does not depolarize L2 cells (Dietl et al. 1995). Hence, it is likely that this Cl- channel is specifically activated by ET by a mechanism which also operates, albeit to a smaller extent, in the absence of an elevation of [Ca2+]i. The finding that intracellular Ca2+ release affects both the magnitude and time course of IET leads to speculation that Cl- channels, stored in intracellular vesicles, might be inserted into the plasma membrane in response to a high threshold [Ca2+]i by vesicle fusion with the plasma membrane.
In summary, the proposed physiological significance of IET is to control non-voltage-gated Ca2+ entry, exerting a negative feedback control on ET-induced Ca2+ signals. The long-lasting activation of this unique Cl- channel could thus interfere with long-term actions of ET, such as cell growth and mitogenesis (Simonson, 1994). In the respiratory epithelium of the lung, the known proliferative effect of ET (Murlas et al. 1995), as well as its effect on transepithelial ion transport, could be modulated in this way. The potential benefit of such a specialized Cl- channel could be that of a specific pharmacological target to control long-term actions of ET.
Acknowledgements
We thank Drs T. Haller, M. Paulmichl and H. Völkl for helpful discussions. The skilful technical assistance of G. Siber and H. Heitzenberger is gratefully acknowledged. This work was supported by the Fonds zur Förderung der Wissenschaftlichen Forschung (FWF), grants P11533-MED and P12974-MED.
Corresponding author
P. Dietl: Department of Physiology, University of Innsbruck, Fritz-Pregl-Strasse 3, A-6020 Innsbruck, Austria.
Email: paul.dietl{at}uibk.ac.at
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Abstract
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Figure 1. The endothelin-1-induced depolarization of the L2 cell membrane: effect of bath ion replacements
) and after (
) the addition of ET (10 nM) in the presence of various bath ion concentrations. Maximum depolarization values were taken. 0 Na+ indicates replacement of bath NaCl by NMDGCl; 0 Ca2+ indicates omission of bath CaCl2. Low Cl- as in A. Each bar represents the mean ±
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Figure 2. The endothelin-1-induced whole-cell current: effect of ion replacement and drugs
) and -65 mV (). Low Cl- and 0 Na+, 0 Ca2+ conditions as described in Fig. 1. 0 K+ indicates replacement of pipette K+ by Cs+. +Ni2+ indicates the presence of 1 mM NiCl2 in the bath, and +Isra the presence of 100 nM (+)-isradipine. Each column represents the mean ±
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Figure 3. The Ca2+ dependence of the endothelin-1-induced whole-cell current
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Figure 4. The effect of 10 nM endothelin-1 (ET) on Sr2+-induced depolarizing spikes
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Figure 5. The effects of mefenamic acid on the endothelin-1-induced whole-cell current and cytoplasmic Ca2+ concentration
) and IET (
). 0 % corresponds to IET and [Ca2+]iET immediately before MFA treatment, values > or < 0 % represent activation or inhibition, respectively, by MFA. For the determination of [Ca2+]iET, only cells with Ca2+ oscillations of < 50 nM amplitude were taken, and an intermediate [Ca2+]i was fitted by eye. Each symbol represents the mean ±
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Figure 6. The effect of low Cl- on the endothelin-1-induced cytoplasmic Ca2+ concentration
DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References
REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References
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