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Cardiovascular Research 1999 44(3):623-631; doi:10.1016/S0008-6363(99)00238-2
© 1999 by European Society of Cardiology
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Copyright © 1999, European Society of Cardiology

Involvement of myosin light-chain kinase in chloride-sensitive Ca2+ influx in porcine aortic endothelial cells

Quang-Kim Trana, Hiroshi Watanabeb,*, Xu-Xia Zhanga, Reiko Takahashia and Ryuzo Ohnoa

aDepartment of Internal Medicine III, Hamamatsu University School of Medicine, 3600 Handa-cho, Hamamatsu 431-3192, Japan
bDepartment of Clinical Pharmacology and Therapeutics, Hamamatsu University School of Medicine, 3600 Handa-cho, Hamamatsu 431-3192, Japan

* Corresponding author. Tel.: +81-53-435-2384; fax: +81-53-435-2384 hwat{at}hama-med.ac.jp

Received 14 April 1999; accepted 15 July 1999


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objectives: This study was designed to investigate the involvement of myosin light-chain kinase (MLCK) in bradykinin- and thapsigargin-induced changes in intracellular Cl– and Ca2+ concentrations ([Cl–]i; [Ca2+]i) in porcine aortic endothelial cells. Methods: Using the fluorescent probes N-ethoxycarbonylmethyl-6-methoxyquinolinium bromide (MQAE) and fura-2/AM, the effects of different MLCK inhibitors on bradykinin- and thapsigargin-induced changes in [Cl–]i and [Ca2+]i were assessed. Results: Bradykinin and thapsigargin significantly decreased the MQAE fluorescence intensity, which indicates increased [Cl–]i; these changes were reversed by removal of extracellular chloride (Cl–o) and were significantly inhibited by Cl–-channel inhibitor N-phenylanthranilic acid but not by Na+–K+–Cl– cotransport inhibitor furosemide. Pretreatment with ML-9 and wortmannin, two different selective inhibitors of MLCK, significantly reduced these changes in a dose-dependent manner. The inhibitory effects of ML-9 and wortmannin on the Cl– responses were not significantly different and were not additive. Bradykinin and thapsigargin provoked large increases in [Ca2+]i, which were significantly diminished by removal of Cl–o and by pretreatment with the Cl–-channel inhibitor N-phenylanthranilic acid. Conclusions: The study shows that an increase in [Cl–]i may be involved in the Ca2+ influx in response to bradykinin and thapsigargin and that MLCK might be involved in the Cl– response. We suggest that MLCK might be involved in the Cl–-sensitive endothelial Ca2+ responses to bradykinin and thapsigargin.

KEYWORDS Endothelial factors; Calcium (cellular)


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Vascular endothelial cells depend on the regulation of intracellular Ca2+ in response to various stimuli for many of their functions. The receptor nonapeptide bradykinin (BK) and the endoplasmic reticulum Ca2+ ATPase inhibitor thapsigargin (TG) have been shown to cause biphasic increases in intracellular Ca2+ concentration. The initial transient increase reflects Ca2+ mobilization from intracellular stores; the sustained component reflects Ca2+ influx from the extracellular space [1–3]; and the so-called capacitative Ca2+ influx hypothesis that internal store depletion triggers the Ca2+ influx has been proposed as the mechanism of this response [4]. However, the precise intracellular events that link the initial store depletion to the subsequent sustained influx await further identification. Ca2+ release from the endoplasmic reticulum as well as Ca2+ influx into endothelial cells has been proven to be regulated by intra- and extracellular concentrations of K+ and Cl– [5–7] and membrane potential [8]. Agonist-induced Ca2+ entry in endothelial cells has also been reported to be Cl–-sensitive [9]. In addition, agonists such as histamine, ATP, and thrombin have been shown to activate intracellular Ca2+ concentration ([Ca2+]i)-dependent Cl– fluxes and Cl– currents with slow activation at positive potentials [10–13]. We have previously documented the essential role of myosin light-chain kinase (MLCK) in Ca2+ signalling in endothelial cells in response to agonist and fluid flow stimulation [14]. In this study, we investigated the changes in intracellular Cl– concentration ([Cl–]i) in response to BK and TG and the role of MLCK in the Cl– responses as well as the relation between the Cl– responses and the Ca2+ responses. The results show that an increase in [Cl–]i may be involved in Ca2+ signalling following stimulation by BK and TG and that MLCK might also have a role in the regulation of the Cl– response.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Cell culture
The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Porcine aortic endothelial cells were isolated and cultured, as previously described [15], by gently scraping the intima of the descending part of porcine aortas. After centrifugation at 250 g for 10 min in Medium 199 (M199; Boehringer, Mannheim, Germany) solution, the pellet of endothelial cells was purified from this suspension, resuspended in M199 solution with Earle's salts, supplemented with 100 IU/ml penicillin G, 100 µM streptomycin, and 20% newborn calf serum (NCS), then aliquoted into polybiphenyl dishes fixed on 10x10-mm glass coverslips, and incubated at 37°C in 5% CO2 for 2 days. The medium was renewed everyday.

2.2 Measurement of [Cl–]i
For assessment of [Cl–]i, the fluorescent dye N-ethoxycarbonylmethyl-6-methoxyquinolinium bromide (MQAE) was used. MQAE, with its high Cl– sensitivity, has been used successfully to measure [Cl–]i in various cell types [16–19]. Cells were loaded with 1 mM MQAE in a solution containing 101 mM Cl– (composition in mM: 5 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid [HEPES], 0.8 MgSO4, 1.0 NaH2PO4, 5.6 glucose, 1.8 Ca acetate, 96 NaCl, 5.3 KCl, 50 mannitol, 22 NaHCO3, pH 7.4) and 10% NCS at pH 7.4 for 2 h at 37°C. The cells were then washed three times with the same solution (without MQAE) to remove MQAE and the serum, and then left unincubated for 10 min before measurements were started. MQAE fluorescence intensity was measured using a fluorescence analyzer (Argus 50, Hamamatsu Photonics, Hamamatsu, Japan) with excitation and emission wavelengths of 360 and 460 nm, respectively. MQAE has a high sensitivity to Cl–, which interacts with and quenches the dye in its excited state; as [Cl–]i increases, MQAE will be quenched out of the cell. Changes in MQAE fluorescence intensity should, therefore, inversely reflect changes in [Cl–]i. BK, TG, furosemide, N-phenylanthranilic acid (NPA), ML-9 and wortmannin had no effect on MQAE fluorescence and autofluorescence of unloaded cells at the concentrations used in this study.

2.3 Measurement of [Ca2+]i
[Ca2+]i was measured in individual endothelial cells as described previously [14,15]. Cells were incubated for 45 min in a modified Tyrode's solution (composition in mM: 150.0 NaCl, 2.7 KCl, 1.2 KH2PO4, 1.2 MgSO4, 1.0 CaCl2 and 10.0 HEPES, with pH 7.4 at 25°C) containing 10% NCS and 2 µM fura-2/AM, a fluorescent Ca2+ indicator. The cells were subsequently washed three times with the modified Tyrode's solution to remove the dye and the serum, and then left unincubated for 20 min before measurements were started. Experiments were performed at 25°C. The fura-2 absorption shift occurring upon binding was determined by scanning the excitation spectra between 340 and 380 nm while monitoring emission at 510 nm. The resultant fluorescent images were analyzed every 30 s from the individual cells with a fluorescence analyzer (Argus 50, Hamamatsu Photonics) using an ultrahigh sensitivity television camera (CCD). The fluorescence ratio (F340/F380) was obtained by dividing, after background subtraction, the 340-nm by the 380-nm images on a pixel-by-pixel basis. Intracellular calibration was performed by the method of Li et al. [20]. To obtain the maximum or minimum value of the fluorescence ratio, after fura-2 loading, endothelial cells were exposed to the modified Tyrode's solution containing 10 µM ionomycin and 3 mM Ca2+ or 5 mM ethylene glycol-bis (beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, respectively. [Ca2+]i was calculated using the equation of Grynkiewics et al. [21]. BK, TG, and NPA had no effect on fura-2 fluorescence or on autofluorescence of unloaded cells at the concentrations used in this study. Cl–-free medium contained (in mM) 5 HEPES, 0.8 MgSO4, 1.0 NaH2PO4, 5.6 glucose, 5.3 KHCO3, 1.8 Ca acetate, 101.3 Na isethionate, 50 mannitol and 16.7 NaHCO3, pH 7.4.

2.4 Materials
M199 was purchased from Boehringer. NCS, penicillin and streptomycin were from Gibco (New York, NY, USA). Fura-2/AM and MQAE were from Dojindo (Kumamoto, Japan). BK, TG, furosemide, NPA and wortmannin were from Sigma (St. Louis, MI, USA). ML-9 was from Calbiochem (La Jolla, CA, USA). BK, TG, NPA, furosemide, ML-9 and wortmannin were dissolved and stored as stock solutions in 1% dimethyl sulfoxide and diluted to the desired concentrations in solutions indicated for each experiment.

2.5 Statistical analysis
Data are expressed as means±SD. Statistical analysis was made using Student's t-test for unpaired data. p<0.01 was considered significant. All experiments were repeated at least three times.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Effects of BK and TG on [Cl–]i in endothelial cells
Endothelial cells were loaded with the fluorescent dye MQAE (1 mM) for 2 h. A typical experiment is shown in Fig. 1. Treatment of the cells with BK (10 µM) and TG (10 µM) at 5 min rapidly caused significant and sustained increases in [Cl–]i, as indicated by reductions in MQAE fluorescence intensity from 12.66±1.86 to 6.66±1.63 and from 12.59±1.23 to 7.06±1.36, respectively (p<0.01) (Fig. 1). As extracellular chloride (Cl–o) was removed, [Cl–]i was gradually reduced to even slightly lower than the pretreated levels, MQAE intensities increasing to 12.83±1.72 and 14.83±1.15, respectively for BK and TG. This was probably due to an [Cl–]o that was lower than the basal levels in the presence of BK and TG. When Cl–o was reintroduced, [Cl–]i concentrations were again significantly quickly increased, MQAE intensities lowered to 7.66±1.36 and 7.29±0.85, respectively (p<0.01) (Fig. 1). The effects of BK and TG on [Cl–]i were dose-dependent, 10 µM provoking the most demonstrable responses, and were not additive (not shown). The reversal of the changes in MQAE intensity as Cl–o was removed confirmed that the changes in MQAE intensity inversely reflect the changes in [Cl–]i in endothelial cells. These results indicate that BK and TG cause Cl– influxes from the extracellular space in endothelial cells.


Figure 1
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  		Fig. 1 Effects of BK and TG on [Cl–]i in endothelial cells in the presence and absence of Cl–o. Endothelial cells were loaded with MQAE (1 mM) for 2 h. The cells were treated with either BK (10 µM) (A) or TG (10 µM) (B) 5 min after measurements were started. Extracellular medium contained 101 mM Cl– until 10 min; Cl–o was then removed until 17 min, when the same concentration of Cl–o was reintroduced. Error bars represent SD; values are means±SD; n=14 cells.

 
3.2 Effects of Na+–K+–Cl– cotransport inhibition and Cl– channel inhibition on the BK- and TG-induced Cl– responses
It has been reported that the Na+–K+–Cl– cotransport is stimulated by agonist stimulation [22,23]. We therefore tested whether the changes in [Cl–]i caused by BK and TG were due to activation of the cotransport alone by using furosemide, an inhibitor of the cotransport [24]. Fig. 2 shows a typical experiment. Pretreatment with furosemide (100 µM) for 5 min did not change the basal MQAE fluorescence intensity, and did not inhibit the changes in [Cl–]i caused by BK and TG; MQAE intensities were reduced from 11.14±1.81 to 4.57±1.95 and from 10.42±1.43 to 3.28±1.20, respectively for BK and TG (p<0.01).


Figure 2
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  		Fig. 2 Effects of the Na+–K+–Cl– inhibitor furosemide on the changes in [Cl–]i caused by BK and TG. Endothelial cells were loaded with 1 mM MQAE for 2 h. Extracellular medium contained 101 mM Cl–. At 5 min after measurements were started, the cells were pretreated with furosemide (100 µM) (arrows). BK (10 µM) (A) and TG (10 µM) (B) were administered at 10 min. Values are means±SD; error bars represent SD; n=14 cells.

 
The Cl– channel inhibitor NPA, however, significantly inhibited the increases in [Cl–]i caused by BK and TG. In the experiment shown in Fig. 3, BK and TG reduced MQAE intensities from 10.57±0.53 to 6.02±0.38, and from 12.20±1.07 to 8.28±1.51, respectively (p<0.01). Pretreatment with NPA (100 µM) did not change the basal intensities, but significantly reduced the changes caused by BK and TG; MQAE intensities were reduced from 10.59±1.42 to only 9.25±1.54 and from 11.83±0.93 to only 10.65±0.89, respectively (p<0.01). There was no significant difference either between the baseline intensities before and after the cells were pretreated with NPA or between the baseline intensities in experiments with and without NPA pretreatment. These results indicate that the changes in [Cl–]i caused by BK and TG were mainly due to activation of NPA-sensitive Cl– channels.


Figure 3
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  		Fig. 3 Effects of the Cl– channel inhibitor NPA on the changes in [Cl–]i caused by BK and TG. Endothelial cells were loaded with 1 mM MQAE for 2 h. Extracellular medium contained 101 mM Cl–. BK (10 µM) (A) and TG (10 µM) (B) were administered at 10 min, with (open circles) or without (closed circles) pretreatment for 5 min with NPA (100 µM). Values are means±SD; error bars represent SD; n=14 cells.

 
3.3 Effects of MLCK inhibitors on BK- and TG-induced changes in [Cl–]i
The effects of MLCK inhibitors on BK and TG-induced increases in [Cl–]i were investigated in endothelial cells loaded with 1 mM MQAE. BK and TG caused rapid increases in [Cl–]i, as indicated by decreases of MQAE fluorescence intensity from 10.57±0.53 to 6.02±0.38, and from 12.20±1.07 to 8.28±1.51, respectively (p<0.01) (Fig. 4A). ML-9 has been used as a specific inhibitor of MLCK [25]; it acts by competing with ATP for binding to the kinase. Pretreatment with ML-9 (100 µM) for 5 min did not change the basal Cl– concentration, but significantly (p<0.01) reduced the changes caused by BK and TG, as indicated by reductions of MQAE intensities from pretreatment levels of 10.83±1.17 and 11.88±1.38 to posttreatment levels of 10.50±0.84 and 10.89±0.76, respectively (Fig. 4A). There was no significant difference either between the baseline intensities before and after pretreatment with ML-9 or between the baseline intensities in experiments with and without ML-9 pretreatment. The effects of ML-9 were dose-dependent at the dose range from 10–100 µM (Fig. 4B). Treatment of the cells with BK (10 µM) and TG (10 µM) significantly reduced MQAE fluorescence intensities by 34.9±5 and 33.5±2.5% of basal levels, respectively. Pretreatment with ML-9 (10–100 µM) for 5 min reduced the responses by BK and TG, at 50 µM the reductions in MQAE intensity being 22.4±2.8 and 22.1±1.9%, and at 100 µM, just 15.1±2.4 and 12.5±2.1% of basal levels, respectively (p<0.01).


Figure 4
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  		Fig. 4 Effects of ML-9 on the Cl– influxes caused by BK and TG. Endothelial cells were loaded with 1 mM MQAE for 2 h. Extracellular medium contained 101 mM Cl–. Panel A: Effects of ML-9 on the Cl– influxes. BK (10 µM) (upper graph) and TG (10 µM) (lower graph) were administered at 10 min, with (open circles) or without (closed circles, taken from Fig. 3) pretreatment with ML-9 (100 µM) (arrows) for 5 min. Values are means±SD; error bars represent SD; n=14 cells. Panel B: Dose-dependent effects of ML-9 on the Cl– influxes. Endothelial cells were pretreated with specified concentrations of ML-9 for 5 min before treatment with BK (10 µM) (upper graph) or TG (10 µM) (lower graph). Values are means±SD and represent the reductions in MQAE fluorescence intensity in comparison with basal levels; error bars represent SD; n=14 cells from each experiments; * represents significant difference from control (p<0.01).

 
To address the issue of specificity for MLCK of the effects seen with ML-9 on the Cl– responses, the effects of wortmannin, a structurally different inhibitor of MLCK [26] were examined (Fig. 5). Wortmannin was shown in our previous work to require at least 30 min to take complete inhibitor effects on agonist-induced Ca2+ influx in endothelial cells [14,15]. Thus, endothelial cells were similarly loaded with MQAE and were pretreated with wortmannin at various doses (10–100 µM) for 30 min. Pretreatment with wortmannin also dose-dependently reduced the responses to BK and TG, at 50 µM the reductions in MQAE intensity being 18.2±2.2 and 22.8±2.9%, and at 100 µM, only 16.5±2.2 and 12.2±2.8% of basal levels, respectively (p<0.01). The effects of ML-9 and wortmannin were not significantly different and were not additive, even at 100 µM.


Figure 5
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  		Fig. 5 Effects of wortmannin on the Cl– influxes caused by BK and TG. Endothelial cells were loaded with 1 mM MQAE for 2 h in the presence of 101 mM Cl–. The cells were then pretreated with specified concentrations of wortmannin for 30 min before treatment with BK (10 µM) (A) or TG (10 µM) (B). Values are means±SD and represent the reductions in MQAE fluorescence intensity in comparison with basal levels; error bars represent SD; n=14 cells from each experiment; * represents significant difference from control (p<0.01).

 
3.4 Dependence of the BK- and TG-induced Ca2+ influxes on [Cl–]o and activation of Cl–channels
Ca2+ influx stimulated by agonists such as histamine, ATP and noradrenaline has been reported to be dependent on [Cl–]i [27,9]. This Ca2+ influx is completely blocked by inhibitors of MLCK [15]. The findings here that the MLCK inhibitors significantly reduced the endothelial Cl– responses to BK and TG also support a link between the Ca2+ and Cl– responses to these agonists. We tested this possibility by first measuring the Ca2+ responses to BK and TG in the presence and absence of extracellular Cl–. BK (10 nM) and TG (1 µM) caused large increases in [Ca2+]i in the presence of Cl–o (modified Tyrode's solution, see Methods) (Fig. 6A). These doses were shown to be enough to provoke demonstrable increases in [Ca2+]i in our previous work [14,15]. Removal of Cl–o (for medium composition, see Methods) significantly, though not completely, inhibited the increases in [Ca2+]i caused by BK and TG (Fig. 6A). In a similar manner, pretreatment of the cells with the Cl– channel inhibitor NPA (100 µM) significantly diminished the BK- and TG-induced increases in [Ca2+] i (Fig. 6B). These results clearly show that the BK- and TG-induced Ca2+ responses are Cl–-dependent and, as MLCK inhibitors have been shown to have complete inhibitor effects on agonist-induced Ca2+ influx in endothelial cells [14,15], these results suggest that MLCK might regulate the Ca2+ influx partly through the regulation of the Cl– influx.


Figure 6
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  		Fig. 6 Dependence of BK- and TG-induced Ca2+ influxes on [Cl–]o and on activation of Cl– channels. Endothelial cells were loaded with 2 µM fura-2/AM for 45 min. Panel A: BK (10 nM) (upper graph) or TG (1 µM) (lower graph) were administered at 10 min. Open circles and closed circles represent the absence and presence of Cl–o (101 mM), respectively. Panel B: Cells were treated with BK (10 nM) (upper graph) or TG (1 µM) (lower graph) with (open circles) or without (closed circles) pretreatment with NPA (100 µM) (arrows) for 5 min. Values are means±SD; error bars represent SD; n=14 cells from each experiment.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
4.1 Mechanisms of the BK- and TG-induced Cl– influx in vascular endothelial cells
Agonist-induced Ca2+ influx in endothelial cells is extremely important for several endothelial functions such as the synthesis and release of vasoactive substances, e.g., nitric oxide, prostaglandin I2 (PGI2), the synthesis of various proteins and gene expressions [28–30]. Previous work has shown that Ca2+ influx provoked by receptor-dependent and independent agonists is Cl– sensitive [27,31,9]. The present study investigated the possibility that the signalling cascade that links the initial Ca2+ release from internal stores and the subsequent Ca2+ influx in response to agonist stimulation in endothelial cells may involve a change in [Cl–]i and examined the possible role of MLCK in the Cl– response.

Various studies have shown that agonists activate Na+–K+–Cl– cotransport in endothelial cells [22,32,23] and that MLCK inhibitors also modulate the cotransport activity [33]. In our study, the increases in [Cl–]i caused by BK and TG, however, were hardly due to activation of the cotransport alone, because the cotransport inhibitor furosemide did not inhibit the changes caused by BK and TG (Fig. 2).

Agonists have also been shown to activate Ca2+-activated chloride channels in endothelial cells [10,34,35]. It is possible in our experiments that the Cl– channels were activated by the increases in [Ca2+]i following the release of internal Ca2+ stores in response to BK and TG, and this activation then contributed to the regulation of the Ca2+ influx. Several lines of evidence support this possibility. First, the fact that NPA, shown to block Ca2+-activated Cl– channels more than other Cl– channels in endothelial cells [35], significantly diminished the changes in [Cl–]i caused by BK and TG (Fig. 3) while furosemide had virtually no effect (Fig. 2) indicates that the Cl– influxes were due to activation of Cl– channels. Second, it has been suggested in endothelial cells that a fast Cl– current is activated secondarily to the release of intracellular Ca2+ stores in response to stimulation with BK [34] and that buffering of intracellular Ca2+ blocked the agonist-induced Ca2+-activated Cl– currents [35]. Third, it is clear that the BK- and TG-induced Ca2+ influx is Cl–-dependent (Fig. 6), while the fact that ML-9 and wortmannin significantly inhibited the BK- and TG-induced increases in [Cl–]i (Fig. 4 and 5Go) indicates that these increases were subsequent to an increase in [Ca2+]i, because MLCK is not activated until [Ca2+]i rises and activates calmodulin. Thus, it is possible that the internal Ca2+ store release caused by BK and TG activates Ca2+-activated Cl– channels, which in turn is involved in the regulation of the Ca2+ influx. The rise in [Ca2+]i due to the influx from extracellular space might then further activate the Cl– influx mechanism.

4.2 Possible involvement of MLCK in BK- and TG-induced Cl– influx
MLCK has been reported to play an important role in the regulation of Ca2+ influx caused by agonist and fluid flow stimulation in endothelial cells [14] and regulate the effects of other agonists on vascular smooth muscle cells [36]. In the present study, ML-9, an MLCK inhibitor, showed significant dose-dependent inhibitor effects on the increases in [Cl–]i caused by BK and TG (Fig. 4A,B). ML-9 is a kinase inhibitor that acts by competing with ATP for binding to the kinase. ML-9 is highly specific for MLCK, but at high doses it can also inhibit protein kinase C (PKC) and cAMP-dependent protein kinase (PKA) [25]. Nevertheless, our previous work has shown that PKA inhibitors have no effects on agonist-induced Ca2+ influx in endothelial cells [15]; and our unpublished data show that bisindolymaleimide I, which strongly inhibits PKC (Ki=10 nM) and PKA at higher doses (Ki=2 µM), significantly increased [Cl–]i, under which circumstance BK and TG had additive effects to increase [Cl–]i. This is supportive of previous findings [23] that PKC activation could exert a negative feedback mechanism on agonist activation of the Na+–K+–Cl– cotransport and further indicates that the effects of BK and TG in our study were not due to activation of the cotransport. More importantly, this negates possible involvement of inhibition of PKC and PKA in the effects seen with ML-9 in our study. In addition, the fact that wortmannin, a structurally unrelated inhibitor of MLCK [26], also inhibited the Cl– responses dose-dependently to the same extent with ML-9 (Fig. 5) further indicates that the inhibitor effects seen with both inhibitors were due to MLCK inhibition. Wortmannin also inhibits PI3 kinase, but the fact that it dose-dependently inhibited both BK- and TG-induced Cl– influx could help rule out phosphatidyl inositol 3-OH (PI3) kinase inhibition as the mechanism responsible for the effects seen, as TG does not activate PI3 kinase.

The mechanisms of the involvement of MLCK in the Cl– influxes, however, were not clear with the present study. MLCK inhibitors might act as inhibitors of Cl– channels or MLCK itself might have some structural link with the Cl– channels that signals their activation when the enzyme is activated. Alternatively, MLCK activation might produce some secondary messenger that is involved in the activation of the Cl– channels. We have previously reported that diphosphorylation of myosin light chain (MLC) by MLCK in response to agonist stimulation in endothelial cells is intimately related to Ca2+ influx [14]. Further study is required to determine if MLCK could be involved the Cl– influx by diphosphorylating MLC, or if the degree of MLC diphosphorylation parallels the involvement of MLCK in agonist-induced increases in [Cl–]i. In addition, although ML-9 and wortmannin exert complete inhibitor effects on the Ca2+ influxes caused by BK and TG [14], their inhibitor effects on the Cl– responses in this study were not complete. Tyrosine kinase has also been shown to regulate TG-induced Cl– influx in another tissue [37]. It is possible, then, that several protein kinases are involved in the regulation of the changes in [Cl–]i in response to agonist stimulation in endothelial cells. Furthermore, the increase in [Cl–]i could only be one of the mechanisms that stimulate Ca2+ influx following the initial Ca2+ release, as removal of Cl–o and pretreatment with NPA did not completely reduce the Ca2+ influxes caused by BK and TG (Fig. 5A,B). Further study is therefore required to quantify the extent to which MLCK is involved in the regulation of the Cl– responses as well as to clarify the mechanism by which it could contribute to the regulation of the Cl– responses.

In conclusion, we have provided evidence that an increase in [Cl–]i may be involved in the Ca2+ signalling cascade that links the internal Ca2+ store release to the Ca2+ influx in endothelial cells in response to BK and TG and that MLCK might be involved in the regulation of the Cl– responses.

Time for primary review 36 days.


   Acknowledgements
 
This study was supported by Grants-in-Aids for Scientific Research from the Ministry of Education, Culture and Science of Japan to H. Watanabe. Quang-Kim Tran is the recipient of a scholarship from the Ministry of Education, Culture and Science of Japan. The authors thank Professor Satoshi Nakamura of Hamamatsu University School of Medicine for his support during the experiments and Aya Takase for technical assistance. The support by The Asian Youth Fellowship Program is greatly appreciated.


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

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