AJP - Heart Calcium Transients and Cell-Sarcomere
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 277: H1498-H1504, 1999;
0363-6135/99 $5.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wang, X.
Right arrow Articles by van Breemen, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wang, X.
Right arrow Articles by van Breemen, C.
Vol. 277, Issue 4, H1498-H1504, October 1999

Depolarization-mediated inhibition of Ca2+ entry in endothelial cells

Xiaodong Wang and Cornelis van Breemen

Vancouver Vascular Biology Research Center and Department of Pharmacology and Therapeutics, Faculty of Medicine, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z3


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The effect of extracellular Cl- in regulating ACh-induced Ca2+ entry into freshly isolated rabbit aortic endothelial cells was studied using Ca2+-sensitive fluorescence microscopy and patch-clamp electrophysiology. After ACh caused transient Ca2+ release in Ca2+-free medium, readdition of 3 mM Ca2+ to the bath maintained Ca2+ entry. Removal of extracellular Cl- abolished the plateau phase in Ca2+ signal and inhibited divalent cation entry. However, in the presence of the K+ ionophore valinomycin, removal of Cl- had no effect on the Ca2+ plateau. Under current-clamp conditions, substitution of gluconate for Cl- induced membrane depolarization. Under voltage clamp, with CsCl in the pipette, ACh activated a slowly developing Cl- current, which was blocked by SITS and 5-nitro-2-(3-phenylpropylamino)benzoic acid. Varying the membrane potential by utilizing different extracellular K+ concentrations in the presence of 5 µM valinomycin demonstrated that depolarization blocked ACh-stimulated Mn2+ entry. These data suggest that ACh-induced Ca2+ entry in freshly isolated endothelial cells requires the presence of extracellular Cl- to maintain a polarized membrane potential.

chloride; receptor-operated channel; calcium influx; endothelium


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ENTRY of Ca2+ from the extracellular space is required for the maintained production of endothelium-derived vasoactive substances (14, 16). The mechanisms whereby agonists induce Ca2+ entry into electrically nonexcitable cells such as endothelium, microglia, and neutrophils remain to be elucidated. Much attention has been focused on an intermediate role of the endoplasmic reticulum (ER) in the activation of this process. According to the "capacitative Ca2+ entry" model, inositol 1,4,5-trisphosphate-mediated ER Ca2+ depletion opens store-operated Ca2+ channels (SOC) (23); however, the search for the link between store depletion and channel activation is not yet completed. We recently showed that, with respect to ACh stimulation of endothelial Ca2+ influx, store depletion is a parallel rather than an obligatory portion of the signal cascade (32). In endothelial cells the same channels may thus be alternatively store operated or receptor operated (ROC).

It is generally accepted that the SOC/ROC are not activated by membrane depolarization (4, 11, 12). On the other hand, there are a number of reports showing that depolarization will drastically inhibit the plateau phase of agonist-stimulated Ca2+ signals in several nonexcitable cell preparations including endothelium (10, 34, 35). In endothelial cells it has been reported that activation of the Ca2+-dependent K+ current leads to membrane hyperpolarization (1, 15, 17, 18, 24, 26, 29, 30). Besides the K+ current, Cl- currents may also play a role in regulating the membrane potential of endothelial cells (5, 7, 33). Nilius and colleagues (19, 21) reported the presence of Ca2+-dependent and/or volume-regulated Cl- channels in several types of vascular endothelial cells. In addition, it has been suggested that Cl- conductance may have modulatory effects on Ca2+ influx (10, 13, 34, 35); for example, in mesangial cells the removal of Cl- from the extracellular space caused immediate abolition of Ca2+ entry (13). The role of Cl- current in regulating Ca2+ homeostasis has not been studied in detail, and little is known about the involvement of Cl- in Ca2+ signaling in endothelial cells.

This study examines the role of extracellular Cl- in the regulation of ACh-stimulated Ca2+ entry in freshly isolated rabbit aortic endothelial cells by using fura 2 fluorescence microscopy and patch-clamp electrophysiology. The data in this communication demonstrate that the ACh-activated Ca2+ entry pathway requires the presence of extracellular Cl- and that a polarized membrane potential may be critical in maintaining Ca2+ influx.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell Isolation

Endothelial cells were freshly isolated from rabbit aorta as described previously (29, 31). Briefly, New Zealand White rabbits were killed after CO2 asphyxiation. The thoracic aorta was removed, cleaned of connective tissue, and placed in physiological saline solution (PSS). After 40 min of enzyme digestion (0.1 mg/ml collagenase, 0.1% elastase, 1 mg/ml trypsin inhibitor) in Ca2+-free PSS at 37°C, endothelial cells were dispersed by titration with the use of a Pasteur pipette and were seeded to glass coverslips precoated with poly-D-lysine. The preparation was kept at 37°C until transferred to the experimental perfusion chamber. The experiments were performed at room temperature.

The final preparation consisted of single cells and small clusters of 3-15 cells that maintained their typical tilelike morphology. Their endothelial nature has been confirmed using 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate-labeled acetylated low-density lipoprotein (DiI-Ac-LDL) uptake assay. Both single cells and the clusters showed rhodamine fluorescence after exposed to DiI-Ac-LDL for 4 h. Either single cells or cell clusters were used for fura 2 fluorescence experiments, and single cells were used for whole cell patch-clamp experiments.

Fura 2 Ca2+ Fluorescence

Intracellular Ca2+ concentration ([Ca2+]i) of the endothelial cells was measured using a microscope-based fluorimeter (Photon Technology International, London, Ontario, Canada). The cells on the coverslip were loaded with 1 µM fura 2-AM for 30 min at 37°C. After 10 min of recovery in dye-free solution, the coverslip was mounted on a Nikon inverted microscope (Nikon Diaphot) with a ×100 oil-immersion quartz objective. The cells were excited alternately at 340 and 380 nm. The emitting fluorescence was collected with a photomultiplier (Hamamatsu Photonics, Shizuoka, Japan) at 510 nm (band-pass filter 20 nm). The ratio of the two intensities at 340- or 380-nm excitation was reported as a relative measure of the free Ca2+ concentration. No calibration was attempted because of the uncertainty of the conventional calibration method in living cells.

Mn2+-quenching experiment. For direct measurement of the divalent cation influx into the endothelial cells, the Mn2+-quenching method was used. The cells were excited at 360 nm, which is the isosbestic point for fura 2 Ca2+-fluorescence. MnCl2 (200 µM) was added to the Ca2+-free bathing solution. After the Mn2+ enters the cells, it binds to the intracellular dye and quenches its fluorescence. The slope of the fluorescence intensity curve gives a measure of the rate of the Mn2+ entry. Only the initial rate, where the Mn2+ quenching curve was linear, was used. After each experiment, 10 µM ionomycin with 0.5 mM MnCl2 was added to the bath to enable maximum quenching. The subsequent steady-state value was taken as the minimum, and the baseline value before MnCl2 addition was taken as the maximum for comparison between different cells.

Electrophysiology

The nystatin-perforated whole cell patch-clamp method was used to study the whole cell current (voltage-clamp mode) and the membrane potential (current-clamp mode) (9). An EPC-7 patch-clamp amplifier (List-electronic, Darmstadt, Germany) and a compatible computer with pCLAMP software (Axon Instruments, Foster City, CA) were used to generate the command pulse and to record data. Continuous data traces were also recorded onto a videotape via a PCM digitizer (Medical Systems) for later analysis using AxoTape software (Axon Instruments).

Patch pipettes were made from borosilicate glass (Warner Instruments, Hamden, CT) with a tip resistance of ~4 MOmega . The pipette was first tip-filled with nystatin-free solution and then backfilled with pipette solution containing 240 µg/ml nystatin. Electrical contact with the cytosol was established in ~15 min after the gigaseal was formed. This was reflected in a decrease in the access resistance below 40 MOmega . To calculate the access resistance, the current trace, generated with a 10-ms voltage pulse of 4 mV (V), was integrated to estimate the total charge (Q), and the time constant (T) was estimated by a exponential fitting to the declining phase of the current. The access resistance (Ra) was calculated using the equations Cm = Q/V and Ra = T/Cm, where Cm is cell membrane capacitance. A high-conductance Agar bridge (1 M KCl) was used in the bath as the ground electrode to minimize the effect of junction potentials caused by solution exchange. All experiments were performed at room temperature.

Solutions

Normal PSS contained (in mM) 126 NaCl, 5 KCl, 1.2 MgCl2, 11 D-glucose, 10 HEPES, and 1 CaCl2, pH 7.4. Ca2+-free PSS contained (in mM) 126 NaCl, 5 KCl, 1.2 MgCl2, 11 D-glucose, and 10 HEPES, pH 7.4. Cl--free solution contained (in mM) 126 Na-gluconate, 5 KCl, 1.2 MgCl2, 11 D-glucose, and 10 HEPES, pH 7.4. The pipette solution contained (in mM) 50 KCl, 85 K-aspartate, 11 EGTA, 1.2 MgCl2, and 10 HEPES, pH 7.2.

In whole cell voltage-clamp experiments, K+ in the pipette was exchanged with Cs+ to eliminate the outward K+ current elicited by ACh. The nystatin pore has a cutoff range of ~200 Da for permitting monovalent ion permeability (8) so that Cs+, which is impermeable to Ca2+-activated K+ channels, can readily pass through. This has been confirmed in whole cell current recording as follows. With the use of a KCl pipette, ACh application induced a transient outward current, which reached a peak current of >1,000 pA within 40 s and then returned toward the baseline within 100 s. In contrast, with the use of a CsCl pipette, only a slow current, which reached a peak current of ~150 pA in ~200 s and was maintained, was seen (holding potential 30 mV).

Materials

5-Nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) was obtained from Research Biochemicals International (Natick, MA). DiI-Ac-LDL was purchased from Biomedical Technologies (Stroughton, MA). Fura 2-AM was from Molecular Probes (Eugene, OR). All other materials were from Sigma Chemical (St. Louis, MO).

Statistics

Results from multiple experiments are presented as means ± SE.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Removal of Extracellular Cl- Abolishes ACh-Induced Ca2+ Entry

Cl- removal abolishes the plateau of the Ca2+ signal induced by ACh. The purpose of the first experiment was to examine the role of extracellular Cl- on ACh-stimulated Ca2+ influx into freshly isolated endothelial cells. Cells seeded on glass coverslips were loaded with fura 2-AM and studied using fluorescence microscopy. At the beginning of the experiment 10 µM ACh was used to deplete the ACh-sensitive store in Ca2+-free PSS. This procedure yielded a transient fura 2 (340/380 nm) signal (Fig. 1) due to Ca2+ release from the ER. After [Ca2+]i returned to baseline, 3 mM Ca2+ was added to the ACh-containing bathing solution, causing a maintained elevation of the fura 2 signal, reflecting stimulated influx of Ca2+ from extracellular space. Substitution of extracellular Cl- with equal molar gluconate totally abolished the plateau of the fura 2 signal.


View larger version (14K):
[in this window]
[in a new window]
  		Fig. 1.   Cl- removal abolishes Ca2+ plateau. Fura 2-AM-loaded endothelial cells were exposed to 10 µM ACh in Ca2+-free (0 Ca2+) physiological saline solution (PSS), which resulted in a transient Ca2+ release. After the intracellular Ca2+ concentration ([Ca2+]i) returned to baseline, 3 mM CaCl2 (3 Ca2+) was added to bath, resulting in a maintained elevation of [Ca2+]i. Removal of extracellular Cl- (substituted by gluconate) abolished this Ca2+ plateau. Data are typical of 5 similar recordings. F340/F380, fura 2 fluorescence as ratio of fluorescence intensity at 340- and 380-nm excitation.

Gluconate substitution inhibits divalent cation entry. To test the hypothesis that the effect of Cl- removal was related to Ca2+ entry, rather than to active Ca2+ transport mediated by the plasma membrane Ca2+-ATPase, Na/Ca2+ exchanger, or sarco(endo)plasmic reticulum Ca2+-ATPase, we used the Mn2+-quenching method to monitor divalent cation entry into fura 2-loaded endothelial cells. Figure 2 shows a representative fura 2 fluorescence recording where endothelial cells were excited at 360 nm in nominally Ca2+-free solution. Addition of 150 µM Mn2+ resulted in a slow decline of the fluorescence, which reflects the basal Mn2+ entry, probably through a nonspecific leak in the cell membrane. Addition of ACh (10 µM) in Na-gluconate-containing solution caused only a minor change in the slope of the fluorescence decay (Fig. 2). This is in strong contrast with the result reported previously in the same cells bathed in normal Cl--containing PSS, in which ACh caused a marked increase in the rate of Mn2+ entry (29, 30). Figure 2 further shows that replenishment of 135 mM Cl- caused an immediate and dramatic increase in Mn2+ quenching for as long as ACh remained present in the bathing solution and until the fura 2 fluorescence had been quenched by ~90%.


View larger version (18K):
[in this window]
[in a new window]
  		Fig. 2.   Cl- removal inhibits divalent cation entry. Fura 2-AM-loaded cells were excited at 360 nm, the isosbestic point of the fura 2 spectra. The rate of decline of fluorescence after Mn2+ addition reflects the rate of divalent cation entry. ACh caused a small increase in Mn2+ entry when cells were bathed in Na-gluconate solution. Substitution of Cl- for gluconate in bath resulted in increased Mn2+ entry, indicating opening of Ca2+-entry channels. Data are typical for 5 similar experiments.

Valinomycin Prevents Inhibition of Ca2+ Entry Caused by Cl- Removal

To test whether the effect of Cl- removal on Ca2+ entry is caused by a change in membrane potential, we used the K+ ionophore valinomycin to clamp the transmembrane potential (Em) close to the K+ equilibrium potential (EK). In Fig. 3 the same experimental protocol represented in Fig. 1 was repeated in the presence of 5 µM valinomycin. In this case Cl- substitution with gluconate failed to reduce the plateau in [Ca2+ ]i. Thus, when the Em was clamped close to EK (see Fig. 6B), Cl- removal did not affect Ca2+ entry. This result suggests that Cl- is not required as a cofactor for Ca2+ transport but is important in regulating the membrane potential after stimulation with ACh.


View larger version (13K):
[in this window]
[in a new window]
  		Fig. 3.   Clamping the membrane potential (Em) obviates Cl- requirement. Cells were exposed to ACh in 0 Ca2+ PSS containing 5 µM valinomycin and fura 2 fluorescence ratio was monitored. Valinomycin, a K+ ionophore, was added to bathing solution to clamp Em close to the equilibrium potential for K+ (EK). After the transient Ca2+ release, 3 mM CaCl2 (3 Ca2+) was added to bath. Under this experimental condition, removal of extracellular Cl- did not affect [Ca2+]i plateau. Data are typical for 4 similar experiments.

Effect of Membrane Depolarization on Stimulated Ca2+ Entry

Valinomycin at different K+ concentrations. The preceding results suggest that removal of Cl- from the extracellular solution blocks stimulation of Ca2+ entry into the endothelial cells and that this effect is caused by a change in Em. To further test the effect of Em on stimulated Mn2+ entry, we used valinomycin to clamp the Em in the presence of different extracellular K+ concentrations ([K+]o). Figure 4A shows representative experiments in which Mn2+ entry was monitored in fura 2-loaded endothelial cells. ACh was applied to the bath when cells were first exposed to a [K+]o of 80 mM (n = 5), 60 mM (n = 5), or 40 mM (n = 6) and then returned to normal PSS with 5 mM [K+]o. Valinomycin (5 mM) was present during the entire protocol.




View larger version (48K):
[in this window]
[in a new window]
  		Fig. 4.   Membrane depolarization inactivates ACh-stimulated divalent cation entry. A: Mn2+ quenching of fura 2 fluorescence in endothelial cells was monitored while 5 µM valinomycin was present in bath. ACh was applied to bathing solution containing different K+ concentrations, and 100 s thereafter K+ concentration of bathing solution was returned to 5 mM. Repolarization after 80 and 60 mM K+, but not after 40 mM K+, caused a marked increase in rate of Mn2+ entry. Y-axis represents normalized fluorescence intensity; curves for 60 and 40 mM K+ were shifted up(down) for clarity. Data are representative traces of multiple experiments. B: relationship between Em and ACh-stimulated divalent cation entry. Initial rate of Mn2+ entry at different extracellular K+ concentrations ([K+]o) relative to rate at [K+]0 = 5 mM (derived from experiments similar to those shown in Fig. 6) were plotted as a function of calculated values of Em. Dashed line represents theoretical effect of electrical driving force on rate of Mn2+ entry (see text for approximation of EMn - EK according to Nernst equation). Driving force at [K+]o = 5 mM was taken as 100%. [K+] for each point is given in parentheses. C: a cell is current-clamped using nystatin-perforated patch. Resting Em is -42 mV. Addition of 5 µM valinomycin hyperpolarized cell to Em of -67 mV. Subsequently, 40, 80, and 131 mM K+ were added to bath, which depolarized cell in a stepwise fashion. Data are typical for 3 similar experiments.

The initial rates of Mn2+ entry at different K+ concentrations were normalized to the rate observed at 5 mM [K+]o in the same cells, and these data are plotted as a function of Em, which was calculated according to the Nernst equation: EK = 58 mV log ([K+]o/[K+]i) (Fig. 4B). The dashed line in Fig. 4B shows the electrical driving force for Mn2+ under the same experimental conditions (EMn - EK), where EMn is the equilibrium potential for Mn2+. For calculation of EMn according to the Nernst equation it was assumed that [Mn2+]i was 100 nM. In reality [Mn2+]i is lower so that the change in driving force for Mn2+ would be less steep than the calculated line in Fig. 4B.

To test the sufficiency of valinomycin in clamping the membrane potential, we recorded Em in endothelial cells using the whole cell (perforated) current clamp. Addition of 5 µM valinomycin in normal PSS (5 mM K+) induced hyperpolarization to -69 ± 3 mV (Fig. 4C). Subsequent changes to concentrations of 40, 80, and 131 mM K+ in the bathing solution caused membrane depolarization (40 mM: -40 ± 6 mV; 80 mM: -18 ± 4 mV; 131 mM: -3 ± 2 mV; no. of experiments for all concentrations = 3). These values were close to the values calculated using the Nernst equation as shown in Fig. 4B.

ACh-Stimulated Whole Cell Cl- Current Contributes to Membrane Potential

Cl- removal causes membrane depolarization after ACh stimulation. Figure 5 shows a record of membrane potential in endothelial cells freshly isolated from rabbit aorta. As reported previously, the resting membrane potential for these cells ranged from -30 to -45 mV (29, 30). At basal condition, Cl- substitution by gluconate caused little change in membrane potential. ACh (10 µM) induced a transient membrane hyperpolarization to about -80 mV due to opening of Ca2+-dependent K+ channels, as reported previously (29). The hyperpolarization caused by ACh declined to near the control level within a few minutes. Subsequent substitution of extracellular Cl- with gluconate caused membrane depolarization from -33 ± 6 mV to -5 ± 3 mV (n = 4).


View larger version (15K):
[in this window]
[in a new window]
  		Fig. 5.   Cl- removal induces depolarization. Em was monitored in perforated whole cell current-clamp mode. Gluconate substitution at resting condition had no significant effect. Addition of 10 µM ACh caused a transient membrane hyperpolarization, after which Em returned to baseline within a few minutes. Substitution of gluconate for extracellular Cl- further depolarized Em. Data are typical for 4 similar experiments.

Whole cell Cl- current induced by ACh. We have previously reported that application of ACh activated a Ca2+-dependent K+ current and caused transient membrane hyperpolarization (29, 30). In this study we tested for the presence of a Cl- current after ACh stimulation. An endothelial cell was voltage clamped at -60 mV using the perforated patch method. Cs+ was included in the pipette instead of K+ to eliminate the ACh-activated K+ current. After ACh application (10 µM), a maintained inward current slowly developed (Fig. 6A). In contrast, when the pipette was filled with KCl, ACh caused a rapid, transient outward current (data not shown). The slow inward current seen after blockade of K+ current returned to baseline on washout of ACh.


View larger version (19K):
[in this window]
[in a new window]
  		Fig. 6.   Whole cell Cl- current activated by ACh. A: an endothelial cell was voltage-clamped at -60 mV using perforated whole cell patch clamp. Pipette solution contained 130 mM CsCl to block Ca2+-dependent K+ current. ACh caused a slowly developing inward current. Trace is representative of >10 experiments. B: current-voltage relationship of ACh-activated current before and after gluconate substitution for Cl- in bath. Currents were measured during application of a voltage ramp from -60 mV to +60 mV. Baseline currents obtained before ACh application were subtracted from both records. C: a cell was voltage clamped at -60 mV using perforated patch containing CsCl in pipette solution. A ramp from -60 mV to +60 mV was applied every 10 s. ACh-activated whole cell current was blocked by 50 µM 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB; n = 4). I, current.

Figure 6B shows the current-voltage (I-V) curves recorded in a cell using a ramp protocol, whereby the cell is depolarized from the holding potential of -60 mV to +60 mV over a time frame of 200 ms. Substitution of gluconate for Cl- in the extracellular solution shifted the reversal potential from -33 mV to ~+32 mV, suggesting that Cl- is the main current carrier. The I-V curves shown were corrected for the leak current measured before ACh application. To confirm that the slow current activated by ACh is a Cl- current, the Cl--channel blockers NPPB and SITS were applied to the extracellular solution. As shown in Fig. 6C, 50 µM NPPB effectively blocked inward and outward currents activated by ACh. The same blocking effect was achieved using 100 µM SITS (data not shown).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

It is generally believed that, like other nonexcitable cells, endothelial cells lack voltage-gated Ca2+ channels (4, 11, 12); instead, the opening of Ca2+-entry channels requires agonist binding to membrane receptors or depletion of intracellular Ca2+ stores. This article reports the modulatory effect of extracellular Cl- and Em on ACh-stimulated Ca2+ entry in freshly isolated endothelial cells. ACh-stimulated Ca2+ entry is abolished on removal of the extracellular Cl-. Furthermore, the data presented here show that the abolition of Ca2+ influx caused by Cl- removal is caused by membrane depolarization based on the presence of a Cl- conductance. These findings suggest that although the activation of Ca2+ entry in endothelial cells is not dependent on voltage, a polarized membrane potential is nevertheless essential in maintaining ACh-induced Ca2+ entry.

Recently, several reports have suggested that extracellular Cl- may play an important role in modulating agonists or store depletion-induced intracellular Ca2+ signaling. Kremer et al. (13) suggested that receptor-activated Ca2+ influx requires the presence of extracellular Cl- as shown in mesangial cells. These authors found that agonist-induced Mn2+ entry was abolished on extracellular substitution of Cl- with gluconate. Similar effects were reported in other cell systems such as rat acinar and human aortic endothelial cells (10, 34, 35).

In our endothelial cell preparation, gluconate substitution abolished the ACh-induced [Ca2+]i plateau and inhibited Mn2+ entry. However, when 5 µM valinomycin was used to clamp the Em, Cl- removal had no effect on the ACh-induced [Ca2+ ]i plateau. Thus Cl- affects Ca2+ entry through its effect on Em, rather than acting as a cofactor for a gating process. Subsequent experiments with different extracellular K+ concentrations in the presence of valinomycin were designed to quantitate depolarization-mediated inhibition of Ca2+ entry channels.

Under normal conditions, 10 µM ACh induced membrane hyperpolarization in endothelial cells due to stimulation of a Ca2+-dependent K+ current as reported previously by us and others (1, 24, 29, 30). We have shown before in this preparation that the hyperpolarization is transient, returning to baseline within a few minutes, and that under certain experimental conditions the ACh-induced response can be maintained after the cells are pretreated with other agonists (30). The maintained hyperpolarization response was strictly dependent on Ca2+ entry from the extracellular space (29, 30). Because endothelial cells lack voltage-activated Ca2+ channels, hyperpolarization, which increases the electrochemical Ca2+ gradient, represents a positive feedback for Ca2+ entry.

Cl- channels have been described in a variety of endothelial cells (5, 7, 19, 21, 33). Using the perforated whole cell patch-clamp, we confirmed in this study the presence of an ACh-stimulated Cl- current in rabbit aortic endothelial cells. Cl- channels may contribute to pH regulation (6) or control of cell proliferation (20, 27, 28), and they obviously contribute to cell membrane potential regulation (27).

Many aspects of the functional role of Cl- channels in intact endothelium are still unknown. ACh-induced responses in endothelial membrane potential have been shown to vary between different preparations. The data could be categorized into two main groups. The first group shows long-lasting agonist-induced membrane hyperpolarization that is mainly carried by K+ current (1-3). In these cases the contribution of Cl- current in maintaining the membrane potential is probably minimal. The second group, which includes rabbit and rat aortic endothelial cells, is characterized by a transient ACh-induced membrane hyperpolarization carried by K+ currents (18, 29, 30). In this case the Cl- current may contribute to the membrane potential after the K+ conductance is largely inactivated. If Cl- and K+ conductance coexist, blockade of the Cl- current will lead to hyperpolarization as reported by Voets et al. (27), and Cl- removal from the extracellular space would lead to depolarization as confirmed in this study.

As can be seen from Fig. 4, in which the rates of Mn2+ entry at different K+ concentrations were compared, depolarization was seen to inhibit Mn2+ entry in a manner that cannot be simply explained as the result of a reduction in the electrical driving force. The inhibition of Mn2+ entry by depolarization was nonlinear and much more pronounced than could be explained by the depolarization-induced decrease in electrochemical driving force (Fig. 4B). Therefore, a more plausible conclusion is that depolarization actually inactivates the Ca2+ entry channel, as is the case for voltage-gated Ca2+ channels.

An alternate explanation for this effect is that the endothelial Ca2+-entry channel displays very strong inward rectification as suggested by Nilius and colleagues (22). They reconstructed a Ca2+ current from the first time derivative of the Ca2+ transient caused by thapsigargin in human umbilical vein endothelial cells and postulated a nonlinear I-V relationship. At this stage, in which no direct single-channel recording of agonist-activated Ca2+ current is available, one cannot distinguish between these two possibilities. However, both explanations suggest that depolarization exerts its effect on Ca2+ entry not merely by decreasing the electrochemical driving force but largely by either inactivation of the channel or the diminishing single-channel conductance. Inactivation of ROC by depolarization is not unique in endothelial cells and has been recently reported by Tabo et al. (25) for smooth muscle.

In conclusion, this study demonstrates that Cl- currents contribute to maintenance of a polarized Em after ACh activation and are critical for the endothelial receptor-mediated Ca2+ influx. This suggests that agonist-induced Ca2+-influx channels in endothelium may possess a voltage-dependent inactivation mechanism like that of voltage-gated Ca2+ channels.


    ACKNOWLEDGEMENTS

This research was supported by a grant from the Medical Research Council of Canada.


    FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: C. van Breemen, Dept. of Pharmacology and Therapeutics, Faculty of Medicine, 2176 Health Sciences Mall, Univ. of British Columbia, Vancouver, BC, Canada V6T 1Z3.

Received 19 January 1999; accepted in final form 3 June 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Busse, R., H. Fichtner, A. Luckhoff, and M. Kohlhardt. Hyperpolarization and increased free calcium in acetylcholine-stimulated endothelial cells. Am. J. Physiol. 255 (Heart Circ. Physiol. 24): H965-H969, 1988[Abstract/Free Full Text].

2.   Chen, G., and D. W. Cheung. Pharmacological distinction of the hyperpolarization response to caffeine and acetylcholine in guinea-pig coronary endothelial cells. Eur. J. Pharmacol. 223: 33-38, 1992[Medline].

3.   Chen, G. F., and D. W. Cheung. Characterization of acetylcholine-induced membrane hyperpolarization in endothelial cells. Circ. Res. 70: 257-263, 1992[Abstract/Free Full Text].

4.   Colden-Stanfield, M., W. P. Schilling, A. K. Ritchie, S. G. Eskin, and L. T. Navarro. Bradykinin-induced increases in cytosolic calcium and ionic currents in cultured bovine aortic endothelial cells. Circ. Res. 61: 632-640, 1987[Abstract/Free Full Text].

5.   Groschner, K., W. F. Graier, and W. R. Kukovetz. Histamine induces K+, Ca2+, and Cl- currents in human vascular endothelial cells. Role of ionic currents in stimulation of nitric oxide biosynthesis. Circ. Res. 75: 304-314, 1994[Abstract/Free Full Text].

6.   Groschner, K., and W. R. Kukovetz. Voltage-sensitive chloride channels of large conductance in the membrane of pig aortic endothelial cells. Pflügers Arch. 421: 209-217, 1992[Medline]. [Corrigenda. Pflügers Arch. 421: September 1992, p. 613.]

7.   Himmel, H. M., A. R. Whorton, and H. C. Strauss. Intracellular calcium, currents, and stimulus-response coupling in endothelial cells. Hypertension 21: 112-127, 1993[Abstract/Free Full Text].

8.   Holz, R., and A. Finkelstein. The water and nonelectrolyte permeability induced in thin lipid membranes by the polyene antibiotics nystatin and amphotericin B. J. Gen. Physiol. 56: 125-145, 1970[Abstract/Free Full Text].

9.   Horn, R., and A. Marty. Muscarinic activation of ionic currents measured by a new whole-cell recording method. J. Gen. Physiol. 92: 145-159, 1988[Abstract/Free Full Text].

10.   Hosoki, E., and T. Iijima. Chloride-sensitive Ca2+ entry by histamine and ATP in human aortic endothelial cells. Eur. J. Pharmacol. 266: 213-218, 1994[Medline].

11.   Jayakody, R. L., C. T. Kappagoda, M. P. Senaratne, and N. Sreeharan. Absence of effect of calcium antagonists on endothelium-dependent relaxation in rabbit aorta. Br. J. Pharmacol. 91: 155-164, 1987[Medline].

12.   Johns, A., T. W. Lategan, N. J. Lodge, U. S. Ryan, C. van Breemen, and D. J. Adams. Calcium entry through receptor-operated channels in bovine pulmonary artery endothelial cells. Tissue Cell 19: 733-745, 1987[Medline].

13.   Kremer, S. G., W. Zeng, R. Hurst, T. Ning, C. Whiteside, and K. L. Skorecki. Chloride is required for receptor-mediated divalent cation entry in mesangial cells. J. Cell. Physiol. 162: 15-25, 1995[Medline].

14.   Kruse, H. J., B. Grunberg, W. Siess, and P. C. Weber. Formation of biologically active autacoids is regulated by calcium influx in endothelial cells. Arterioscler. Thromb. 14: 1821-1828, 1994[Abstract/Free Full Text].

15.   Laskey, R. E., D. J. Adams, A. Johns, G. M. Rubanyi, and C. van Breemen. Membrane potential and Na+-K+ pump activity modulate resting and bradykinin-stimulated changes in cytosolic free calcium in cultured endothelial cells from bovine atria. J. Biol. Chem. 265: 2613-2619, 1990[Abstract/Free Full Text].

16.   Luckhoff, A., U. Pohl, A. Mulsch, and R. Busse. Differential role of extra- and intracellular calcium in the release of EDRF and prostacyclin from cultured endothelial cells. Br. J. Pharmacol. 95: 189-196, 1988[Medline].

17.   Marchenko, S. M., and S. O. Sage. Calcium-activated potassium channels in the endothelium of intact rat aorta. J. Physiol. (Lond.) 492: 53-60, 1996[Medline].

18.   Marchenko, S. M., and S. O. Sage. Mechanism of acetylcholine action on membrane potential of endothelium of intact rat aorta. Am. J. Physiol. 266 (Heart Circ. Physiol. 35): H2388-H2395, 1994[Abstract/Free Full Text].

19.   Nilius, B., J. Eggermont, T. Voets, and G. Droogmans. Volume-activated Cl- channels. Gen. Pharmacol. 27: 1131-1140, 1996[Medline].

20.   Nilius, B., J. Prenen, M. Kamouchi, F. Viana, T. Voets, and G. Droogmans. Inhibition by mibefradil, a novel calcium channel antagonist, of Ca2+- and volume-activated Cl- channels in macrovascular endothelial cells. Br. J. Pharmacol. 121: 547-555, 1997[Medline].

21.   Nilius, B., G. Szucs, S. Heinke, T. Voets, and G. Droogmans. Multiple types of chloride channels in bovine pulmonary artery endothelial cells. J. Vasc. Res. 34: 220-228, 1997[Medline].

22.   Oike, M., M. Gericke, G. Droogmans, and B. Nilius. Calcium entry activated by store depletion in human umbilical vein endothelial cells. Cell Calcium 16: 367-376, 1994[Medline].

23.   Putney, J. W., Jr. The capacitative model for receptor-activated calcium entry. Adv. Pharmacol. 22: 251-269, 1991.

24.   Sakai, T. Acetylcholine induces Ca-dependent K currents in rabbit endothelial cells. Jpn. J. Pharmacol. 53: 235-246, 1990[Medline].

25.   Tabo, M., T. Ohta, S. Ito, and Y. Nakazato. Effects of external K+ on depletion-induced Ca2+ entry in rat ileal smooth muscle. Eur. J. Pharmacol. 313: 151-158, 1996[Medline].

26.   Usachev, Y. M., S. M. Marchenko, and S. O. Sage. Cytosolic calcium concentration in resting and stimulated endothelium of excised intact rat aorta. J. Physiol. (Lond.) 489: 309-317, 1995[Medline].

27.   Voets, T., G. Droogmans, and B. Nilius. Membrane currents and the resting membrane potential in cultured bovine pulmonary artery endothelial cells. J. Physiol. (Lond.) 497: 95-107, 1996[Medline].

28.   Voets, T., G. Szucs, G. Droogmans, and B. Nilius. Blockers of volume-activated Cl- currents inhibit endothelial cell proliferation. Pflügers Arch. 431: 132-134, 1995[Medline].

29.   Wang, X., W. Chu, F. Lau, and C. van Breemen. Bradykinin potentiates acetylcholine induced responses in native endothelial cells from rabbit aorta. Biochem. Biophys. Res. Commun. 213: 1061-1067, 1995[Medline].

30.   Wang, X., W. Chu, and C. van Breemen. Potentiation of acetylcholine-induced responses in freshly isolated rabbit aortic endothelial cells. J. Vasc. Res. 33: 414-424, 1996[Medline].

31.   Wang, X., F. Lau, L. Li, A. Yoshikawa, and C. van Breemen. Acetylcholine-sensitive intracellular Ca2+ store in fresh endothelial cells and evidence for ryanodine receptors. Circ. Res. 77: 37-42, 1995[Abstract/Free Full Text].

32.   Wang, X., and C. van Breemen. Multiple mechanisms of activating Ca2+ entry in freshly isolated rabbit aortic endothelial cells. J. Vasc. Res. 34: 196-207, 1997[Medline].

33.   Watanabe, M., K. Yumoto, and R. Ochi. Indirect activation by internal calcium of chloride channels in endothelial cells. Jpn. J. Physiol. 4, Suppl. 2: S233-S236, 1994.

34.   Yumoto, K., H. Yamaguchi, and R. Ochi. Depression of ATP-induced Ca2+ signalling by high K+ and low Cl- media in human aortic endothelial cells. Jpn. J. Physiol. 45: 111-122, 1995[Medline].

35.   Zhang, G. H., and J. E. Melvin. Membrane potential regulates Ca2+ uptake and inositol phosphate generation in rat sublingual mucous acini. Cell Calcium 14: 551-562, 1993[Medline].

Am J Physiol Heart Circ Physiol 277(4):H1498-H1504
0002-9513/99 $5.00 Copyright © 1999 the American Physiological Society


This article has been cited by other articles:


Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
M. L. Paffett, J. S. Naik, T. C. Resta, and B. R. Walker
Reduced store-operated Ca2+ entry in pulmonary endothelial cells from chronically hypoxic rats
Am J Physiol Lung Cell Mol Physiol, November 1, 2007; 293(5): L1135 - L1142.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
X. F. Figueroa, C.-C. Chen, K. P. Campbell, D. N. Damon, K. H. Day, S. Ramos, and B. R. Duling
Are voltage-dependent ion channels involved in the endothelial cell control of vasomotor tone?
Am J Physiol Heart Circ Physiol, September 1, 2007; 293(3): H1371 - H1383.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
J.-Z. Sheng and A. P. Braun
Small- and intermediate-conductance Ca2+-activated K+ channels directly control agonist-evoked nitric oxide synthesis in human vascular endothelial cells
Am J Physiol Cell Physiol, July 1, 2007; 293(1): C458 - C467.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
G. H. Seol, S. C. Ahn, J. A. Kim, B. Nilius, and S. H. Suh
Inhibition of endothelium-dependent vasorelaxation by extracellular K+: a novel controlling signal for vascular contractility
Am J Physiol Heart Circ Physiol, January 1, 2004; 286(1): H329 - H339.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
S. P. Marrelli, M. S. Eckmann, and M. S. Hunte
Role of endothelial intermediate conductance KCa channels in cerebral EDHF-mediated dilations
Am J Physiol Heart Circ Physiol, October 1, 2003; 285(4): H1590 - H1599.
[Abstract] [Full Text] [PDF]

Home page
 Physiol. Rev.Home page
B. Nilius and G. Droogmans
Ion Channels and Their Functional Role in Vascular Endothelium
Physiol Rev, October 1, 2001; 81(4): 1415 - 1459.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
J. A. Rosado and S. O. Sage
Role of the ERK Pathway in the Activation of Store-mediated Calcium Entry in Human Platelets
J. Biol. Chem., May 4, 2001; 276(19): 15659 - 15665.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wang, X.
Right arrow Articles by van Breemen, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wang, X.
Right arrow Articles by van Breemen, C.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online