Mechanism of endothelin-1-(1---31)-induced calcium signaling in human coronary artery smooth muscle cells

Daisuke Inui1, Masanori Yoshizumi1, Naoko Okishima2, Hitoshi Houchi1, Koichiro Tsuchiya1, Hiroshi Kido2, and Toshiaki Tamaki1

1 Department of Pharmacology and 2 Division of Enzyme Chemistry, The University of Tokushima School of Medicine, Tokushima 770-8503, Japan


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We have found that human chymase produces a 31-amino acid endothelin [ET-1-(1---31)] from the 38-amino acid precursor (Big ET-1). We examined the mechanism of synthetic ET-1-(1---31)-induced intracellular Ca2+ signaling in cultured human coronary artery smooth muscle cells. ET-1-(1---31) increased the intracellular free Ca2+ concentration ([Ca2+]i) in a concentration-dependent manner (10-14-10-10 M). This ET-1-(1---31)-induced [Ca2+]i increase was not affected by phosphoramidon, Bowman-Birk inhibitor, and thiorphan. The ET-1-(1---31)-induced [Ca2+]i increase was not influenced by removal of extracellular Ca2+ but was inhibited by thapsigargin. ET-1-(1---31) at 10-12 M did not cause Ca2+ influx, whereas 10-7 M ET-1-(1---31) evoked marked Ca2+ influx, which was inhibited by nifedipine. ET-1-(1---31) also increased inositol trisphosphate formation. These results suggest that the ET-1-(1---31)-induced [Ca2+]i increase at relatively low concentrations is attributable to the release of Ca2+ from inositol trisphosphate-sensitive intracellular stores, whereas Ca2+ influx into the cells evoked by high concentration of ET-1-(1---31) probably occurs through voltage-dependent Ca2+ channels. We concluded that the physiological activity of ET-1-(1---31) may be attributable to Ca2+ mobilization from intracellular stores rather than influx of Ca2+ from extracellular space.

human chymase; confocal laser microscopy


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

ENDOTHELIN-1 (ET-1) is a 21-amino acid polypeptide that exhibits various physiological actions, such as vascular contraction (40), cardiac hypertrophy, and mitogenesis (2). Human ET-1 is generated from the 38-amino acid precursor (Big ET-1), through a cleavage of the Trp21-Val22 bond via the action of endothelin (ET)-converting enzyme (ECE). Although ECE was originally shown to be a membrane-bound metalloprotease (40), several other metalloproteases have also been shown to catalyze the formation of ET-1 from Big ET-1. For example, an aspartic protease (7, 35), a metalloendopeptidase (19, 22), cathepsin D-like enzyme (27), and elastase (10) have been identified as putative converters of Big ET-1 to ET-1. Rat mast cell chymase has also been reported as a putative converter of Big ET-1 to ET-1 (36).

We have recently reported that human mast cell chymase, unlike rat mast cell chymases, selectively cleaves Big ET-1 at the Tyr31-Gly32 bond to produce novel trachea-constricting 31-amino acid endothelins, ET-1-(1---31), without any further degradation products (18). ET-1-(1---31) may potentially be produced in the lung under certain circumstances, because in vitro study with human chymase, which is an alternative angiotensin-converting enzyme in human tissue (32), has shown that Big ET-1 can be converted to ET-1-(1---31). Because ET-1 has been shown to play a significant role in human cardiovascular functions (2), ET-1-(1---31) may possess biological activities as well in human tissues. As it has been reported that chymase plays a significant role in the foam cell formation in human coronary atheromas (14), ET-1-(1---31) may be an atherogenetic substance in human tissues.

Recently, we have found that synthetic ET-1-(1---31) induces a rise in intracellular free calcium concentration ([Ca2+]i) in cultured human coronary artery smooth muscle cells (41). We also observed that ET-1-(1---31) causes a contraction of microperfused rabbit afferent and efferent arterioles (29). ET-1 has been shown to affect voltage-dependent Ca2+ channels and to cause Ca2+ influx into vascular smooth muscle cells (3, 28). On the other hand, it has been reported that ET-1 activates phospholipase C, which results in Ca2+ mobilization from intracellular stores (15, 17, 33). Therefore, we investigated in this study the mechanism of ET-1-(1---31)-induced Ca2+ signaling with cultured human coronary artery smooth muscle cells. Change in [Ca2+]i elicited by synthetic ET-1-(1---31) was measured by confocal laser microscopy. The ET-1-(1---31)-induced Ca2+ influx into the cells was also examined with 45CaCl2. In addition, the effects of known ET-receptor antagonists and various protease inhibitors on change in ET-1-(1---31)-induced [Ca2+]i were examined.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Cell preparation and culture. Human coronary artery smooth muscle cells were purchased from Clonetics (San Diego, CA). Cells were plated in 25-cm2 tissue culture flasks at a density of 5 × 103 cells/cm2 in MCDB-131 medium supplemented with 5% heat-inactivated fetal calf serum, 0.5 ng/ml epidermal growth factor, 1 ng/ml basic fibroblast growth factor, 5 µg/ml insulin, 50 µg/ml gentamicin, and 0.25 µg/ml amphotericin B. The cells were incubated at 37°C in 5% CO2, and the medium was replaced every other day until 60-80% confluent. Then, the cells were removed from the flasks with 0.025% trypsin plus 0.01% EDTA and washed twice with HEPES buffer solution (in mM: 30 HEPES, 130 NaCl, 3.0 KCl, 3.0 Na2HPO4, and 10 glucose, adjusted with NaOH to pH 7.40). Thereafter, the cells were seeded onto glass coverslips attached to 35-mm tissue culture dishes coated with poly-L-lysine. The dishes were purchased from MatTek (Ashland, MA). All experiments were performed with the cells in passages 5-15 and at 2-3 days postconfluence.

Loading of the Ca2+ indicator fluo 3 into cells. The culture medium was removed from the dishes and replaced with modified Krebs-Henseleit bicarbonate buffer solution (K-H solution; in mM: 135 NaCl, 5.6 KCl, 1.2 MgSO4, 1.2 KH2PO4, 25 NaHCO3, 2.2 CaCl2, and 10 glucose, adjusted with HCl to pH 7.40) oxygenated with a 95% O2-5% CO2 gas mixture. Then, the cells were loaded with the ester form of the dye (fluo 3-acetoxymethyl ester). For this purpose, the cells were incubated at 37°C with a final concentration of 4 µM fluo 3-acetoxymethyl ester. After a loading period of 30 min, the solution was exchanged with a dye-free K-H solution, and the cells were allowed to deesterify the indicator for an additional 10 min. The low ester concentration was chosen to minimize problems arising from compartmentalization of the indicator (9).

Measurement of fluorescence intensity by confocal laser microscopy. We used the methods for confocal fluorescence measurements that have been reported previously (30). The confocal imaging system (RCM 8000, Nikon, Tokyo, Japan) with an argon-ion laser was attached to an inverted microscope (Nikon TMD300, Diaphot, Tokyo, Japan). Cells in the culture dish containing 1 ml of K-H solution were placed on the stage of the microscope, and the fluorescence in the cell was excited at 488 nm by the laser. Emission at wavelengths longer than 520 nm was then detected by a photomultiplier. The system scanned full-field images at 30 frames/s, but the images were obtained by averaging eight successive frames in order to improve the signal-to-noise ratio. The objective lens used was a Nikon CF Fluor ×20/NA0.75. After stable baseline fluorescence intensity was measured, 10 µl of an agent was added to extracellular medium to yield a 1/100 concentration, and the fluorescence intensity was recorded. The same cells were stimulated by 10 µM of ionomycin 1 min after test agent application, and the relative fluorescence intensity was calculated. For experiments of the inhibition of Ca2+ release from intracellular stores, thapsigargin, an inhibitor of sarcoplasmic reticulum Ca2+-ATPase, was added during dye loading. These experiments were performed under Ca2+-free conditions. Calibration of the fluo 3 fluorescence intensity to estimate [Ca2+]i was calculated from the difference between Fmax and Fmin. Fmin was defined as the minimum fluorescence intensity before each agonist stimulation. Fmax was estimated from the intensity at 1 min after addition of ionomycin in each experiment. Results were expressed as percentages of the difference between Fmax and Fmin. For data analysis, the images of confocal microscopic observation were stored on a Panasonic magnetico-optical disk (Matsushita Electric Industrial, Osaka, Japan). Sequences of digitized images were transferred to an IBM-Think Pad equipped with an image-processing software package. Data are presented as means ± SE.

Measurement of Ca2+ influx into the cells. The method for measurement of 45Ca2+ influx into the cells was as described previously (42). Briefly, the cultured cells were washed once with 1 ml of K-H solution and then incubated with test agents at 37°C for 10 min in K-H solution containing 45CaCl2 (4 µCi/ml). Then, the medium was discarded and the cells were washed three times with 1 ml of ice-cold Ca2+-free K-H solution. The cells were then solubilized by 1% Triton X-100, and the radioactivity in the cell lysate was measured in a liquid scintillation counter. The amount of 45Ca2+ taken up into the cells was calculated on the basis of the specific activities of radioactive 45Ca2+ in the reaction mixture and expressed in nanomoles/dish (1 × 106 cells). For experiments of the inhibition of Ca2+ influx into the cells, nifedipine, a typical voltage-dependent Ca2+ channel blocker, was added to the medium during incubation.

Measurement of inositol trisphosphate formation within the cells. Agonist-induced inositol trisphosphate (IP3) formation within the cells was measured with a 3H assay system kit (Amersham, Tokyo, Japan; Ref. 21). Briefly, the cultured cells were washed once with 1 ml of K-H solution and then incubated at 37°C for 15 s with or without test agents, and the reaction was stopped by liquid nitrogen freezing. For experiments with ET-receptor subtype (ETA; ETB) antagonists, a selective ETA antagonist, BQ123 (6), or a selective ETB antagonist, BQ788 (8), was added to the medium during incubation. Then, both the medium and cell lysate were centrifuged at 2,000 g for 5 min. The resultant supernatant was further concentrated five times with a Speed-Vac concentrator (SVC 100, Savant, NY) and was stored frozen at -70°C until assayed. The IP3 in the supernatant was assayed with a D-myo-[3H]inositol 1,4,5-trisphosphate assay system (7.5 nCi/ml). Results were expressed as picomoles per dish (1 × 106 cells).

Statistics. One-way ANOVA was used to determine the significance among groups, after which the modified t-test with the Bonferroni correction was used for comparison between individual groups. A value of P < 0.05 was considered to be statistically significant.

Materials. Human ET-1, Big ET-1, and phosphoramidon were obtained from Peptide Institute (Osaka, Japan). ET-1-(1---31) was synthesized by solid-phase procedures by the Peptide Institute. MCDB-131 medium, fetal calf serum, epidermal growth factor, insulin, gentamicin, amphotericin B, and trypsin were obtained from Clonetics. The D-myo-[3H]inositol 1,4,5-trisphosphate assay system and 45CaCl2 were obtained from Amersham. Fluo 3-acetoxymethyl ester, ionomycin, and Triton X-100 were purchased from Wako Pure Chemical (Osaka, Japan). BQ123 and BQ788 were gifts from Banyu Pharmaceutical (Tsukuba, Japan). All other chemicals, including Bowman-Birk inhibitor (BBI), thapsigargin, and nifedipine were from Sigma (St. Louis, MO).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Concentration-dependent increase in [Ca2+]i induced by ET-1-(1---31). Figure 1 shows the time course of 10-12 M ET-1-(1---31)-induced increase in [Ca2+]i. The ET-1-(1---31)-induced increase in [Ca2+]i reached a peak within 10 s and then gradually decreased to the baseline resting value within 1 min. ET-1-(1---31) caused an increase in [Ca2+]i in a concentration-dependent manner from 10-14 M to 10-10 M. ET-1 also increased [Ca2+]i; however, it was ~10× more potent than ET-1-(1---31) at the concentrations used. The concentration-response curve for Big ET-1 was similar to that for ET-1-(1---31) (41). However, the concentrations of ET-1-(1---31) that caused half-maximal increases in [Ca2+]i were ~100× and 10,000× lower than those of angiotensin II and norepinephrine, respectively (11).


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Fig. 1.   Typical time course of 31-amino acid endothelin [ET-1-(1---31)]-induced increase in intracellular free Ca2+ concentration ([Ca2+]i) in cultured human coronary artery smooth muscle cells. Cells were stimulated with 10-12 M ET-1-(1---31), followed by addition of 10 µM ionomycin 1 min later. Values are expressed as percent difference of Fmax and Fmin as described in text. Fmax, fluorescence intensity on addition of 10 µM ionomicin to incubation medium; Fmin, minimum fluorescence intensity before each agonist stimulation.

Possible conversion of ET-1-(1---31) to ET-1 by ECE. To investigate the possibility that the ET-1-(1---31)-induced increase in [Ca2+]i is due to the degradation of ET-1-(1---31) to ET-1 by ECE in the incubation medium or the cells, we examined the effects of an ECE inhibitor and protease inhibitors on the increase. As shown in Table 1, phosphoramidon, a potent inhibitor of ECE (16), at 10-5 and 10-4 M did not inhibit the increase in [Ca2+]i caused by 10-12 M ET-1-(1---31). Similar results were obtained when the cells were preincubated with BBI and thiorphan, known inhibitors of trypsin- or chymotrypsin-type proteases and neutral endopeptidase 24.11 (22), in the medium at concentrations of 10-5 M. Phosphoramidon at 10-4 M reduced the increase in [Ca2+]i caused by 10-12 M Big ET-1.

                              
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Table 1.   Effects of phosphoramidon, BBI, and thiorphan on ET-1-(1---31)-induced increase in [Ca2+]i in cultured human coronary artery smooth muscle cells

Ca2+ mobilization from intracellular stores and from the extracellular space. Figure 2 shows the responses of cultured coronary artery smooth muscle cells to 10-12 M ET-1-(1---31) in the presence or absence of extracellular Ca2+. ET-1-(1---31)-induced increase in [Ca2+]i was not affected by removal of Ca2+ from the medium. However, it was almost abolished by thapsigargin (10-5 M), a specific inhibitor of the sarcoplasmic reticulum Ca2+ pump. From these results, it is assumed that a 10-12 M ET-1-(1---31)-induced increase in [Ca2+]i is attributable to the release of Ca2+ from intracellular stores. On the other hand, ET-1-(1---31) at 10-7 M caused marked 45Ca2+ influx into the cells, as did ET-1 (10-7 M) and Big ET-1 (10-7 M; Table 2). The 10-7 M ET-1-(1---31)-induced 45Ca2+ influx was almost abolished by the addition of 10-5 M nifedipine, a voltage-dependent Ca2+ channel blocker, and nifedipine also abolished the Ca2+ influx induced by ET-1 and Big ET-1. ET-1-(1---31) at 10-12 M did not evoke Ca2+ influx from the extracellular space nor did ET-1 (10-12 M) and Big ET-1 (10-12 M).


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Fig. 2.   Effect of extracellular Ca2+ removal on ET-1-(1---31)-induced rise in [Ca2+]i and its inhibition by thapsigargin (triangle ), a specific inhibitor of sarcoplasmic Ca2+ pump. ET-1-(1---31) (10-12 M)-induced change in [Ca2+]i was measured in presence (open circle ) or absence () of extracellular Ca2+. Thapsigargin (10-5 M) was added to incubation medium throughout dye loading and experiments, which were performed under Ca2+-free conditions (triangle ). Values are expressed as percent difference as described in text (means of 5 separate experiments). Maximal SE was 7% of Fmax - Fmin. To avoid complicating figures, error bars were omitted. Peak level with thapsigargin in Ca2+-free medium was significantly less than value evoked by ET-1-(1---31) in normal medium (P < 0.05).


                              
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Table 2.   Effects of low (10-12 M) and high (10-7 M) doses of ET-1-(1---31), ET-1, and Big ET-1 on 45Ca2+ influx into cells

IP3 formation within the cells induced by ET-1-(1---31). As shown in Fig. 3, application of 10-10 M ET-1-(1---31) caused an ~3.2-fold increase of IP3 formation from the control value during 15 s of incubation. The ET-1-induced IP3 level was similar to that of ET-1-(1---31), whereas Big ET-1 was less potent than ET-1-(1---31). Next, we examined the effects of known ET-receptor antagonists. The ET-1-(1---31)-induced IP3 formation was also inhibited by the selective ETA antagonist BQ123 (10-8 M) but not by the selective ETB antagonist BQ788 (10-8 M).


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Fig. 3.   Effects of ET-1, ET-1-(1---31), and 38-amino acid precursor (Big ET-1) on inositol trisphosphate formation within cells. Cells were incubated with each agent at 10-10 M for 15 s. ET antagonists BQ123 and BQ788 at 10-8 M were added during incubation period. Accumulation of D-myo-[3H]inositol 1,4,5-trisphosphate was assayed as described in METHODS. Data are means ± SE of 5 separate experiments. * Statistical significance from value induced by ET-1-(1---31) (P < 0.05).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The present study demonstrated that ET-1-(1---31), which is cleaved at the Tyr31-Gly32 bond of Big ET-1 by human chymase, caused a rise in [Ca2+]i in cultured human coronary artery smooth muscle cells. The ET-1-(1---31)-induced rise in [Ca2+]i at relatively low concentrations may be attributable to the mobilization of Ca2+ from intracellular stores, whereas ET-1-(1---31) causes marked Ca2+ influx into the cells at high concentrations around the nanomolar range. We also demonstrated that the ET-1-(1---31)-induced phenomena are mediated through ETA or ETA-like receptors of the cells, which may be coupled to the IP3-forming pathway.

Human chymase is highly efficient in converting angiotensin I to angiotensin II (32). Urata et al. (31) reported that the chymase-dependent angiotensin II-forming pathway is a major pathway for angiotensin II formation in the failing human heart. We have recently found that human mast cell chymase specifically converted Big ET-1 to the novel trachea-constricting 31-amino acid peptide ET-1-(1---31), which is different in amino acid length from the well-known 21-amino acid ET-1 (18). It has also been reported that a serine protease in the membrane fraction of human lung hydrolyzes Big ET-1 to ET-1-(1---31), which showed contractile activity in the pulmonary artery (4). Because ET-1 as well as angiotensin II has been shown to possess a wide variety of biological actions contributing to vascular contraction, cardiac hypertrophy, or atherosclerosis (1, 2), ET-1-(1---31) may also be a novel and alternative vasoactive peptide in the ET family. Recently, we found that ET-1-(1---31) causes [Ca2+]i increase in cultured human coronary artery smooth muscle cells (41). We also observed that ET-1-(1---31) induces contraction of rabbit afferent and efferent arterioles, porcine coronary arteries, and rat aortae (12, 29). Because the increase in [Ca2+]i caused by ET-1 has been found to play a role in vascular contraction or vascular smooth muscle cell proliferation (13), ET-1-(1---31) is an alternative candidate for the causative substance for these events in vivo. In the present study, we investigated the mechanism of Ca2+ signaling elicited by synthetic ET-1-(1---31) with cultured human coronary artery smooth muscle cells.

The results shown in Fig. 1 revealed that ET-1-(1---31) in the picomolar range causes a transient increase in [Ca2+]i in cultured human coronary artery smooth muscle cells. The effect of Big ET-1 in vivo is comparable with that of ET-1, which is likely due to the conversion of Big ET-1 to ET-1 (16). Therefore, it is important to elucidate whether the response to ET-1-(1---31) is mediated by ET-1 after hydrolysis of ET-1-(1---31) or whether ET-1-(1---31) itself acts directly on the receptors of the cells. As shown in Table 1, phosphoramidon, an inhibitor of metalloendopeptidases and ECE (16), had almost no effect on the increase in [Ca2+]i elicited by ET-1-(1---31). BBI and thiorphan, which are inhibitors of trypsin- or chymotrypsin-type proteases and neutral endopeptidase 24.11 (18, 37), both failed to inhibit the ET-1-(1---31)-induced [Ca2+]i increase. However, the Big ET-1-induced increase in [Ca2+]i was suppressed by phosphoramidon (Table 1). These results suggest that the ability of ET-1-(1---31) to increase [Ca2+]i is not the consequence of its conversion to ET-1 by ECE, a chymotrypsin-type protease(s), or metalloendopeptidase(s). Moreover, it is reported that ET-1-(1---31) is not a substrate for ECE purified from bovine adrenal cortex (37). Nevertheless, it should be noted that ET-1-(1---31) itself has biological activity in cultured human coronary artery smooth muscle cells. Moreover, in a preliminary study, we have detected ET-1-(1---31) not only in human lungs but also in human hearts, and the amount of ET-1-(1---31) in autopsy specimens was similar to or higher than that of ET-1. ET-1-(1---31) is thus a novel putative vasoactive peptide member of the ET family and may play a significant role in chymase-related pathophysiological processes in humans.

It has been reported that there are at least two main subtypes of ET-receptors, termed ETA and ETB (34). The ETA receptor was originally found in vascular smooth muscle cells (26), and recent studies revealed that ETB receptors also exist in vascular smooth muscle cells (24). These have been called ETB2 receptors to distinguish them from the ETB1 vasodilatory receptors (20). In our study, the ET-1-(1---31)-induced increase in [Ca2+]i was inhibited by BQ123, but not by BQ788, known inhibitors of ETA and ETB receptors, respectively (41). Although the vasoconstrictive actions of ET-1-(1---31) are blocked by ETA-specific antagonists, it does not necessarily mean that there are not specific receptor subtypes through which these effects are mediated. Our results suggest that the ET-1-(1---31)-induced response of the cells is mediated through ETA or ETA-like receptors. Further studies are needed to clarify whether different receptors are present.

In this study, we investigated the characteristics of Ca2+ signaling elicited by ET-1-(1---31) in human coronary artery smooth muscle cells. As shown in Fig. 2, the 10-12 M ET-1-(1---31)-induced increase in [Ca2+]i was not affected by removal of extracellular Ca2+ but was inhibited by thapsigargin, a specific inhibitor of the sarcoplasmic reticulum Ca2+ pump. The [Ca2+]i increase evoked by ET-1 at 10-13 M was also not affected by removal of extracellular Ca2+ but was inhibited by thapsigargin (data not shown). From these results, it is concluded that ET-1-(1---31) in the picomolar range causes Ca2+ mobilization from intracellular stores. ET-1 induces rapid and transient increase in [Ca2+]i, probably through the activation of IP3 formation, in vascular smooth muscle cells (38, 39). We also observed increase in IP3 formation after ET-1-(1---31) treatment (Fig. 3). However, the above studies that examined IP3 formation have used relatively high concentrations (nM range) of ET-1, and at these concentrations, significant Ca2+ influx after the initial increase of [Ca2+]i may occur. Our results shown in Table 2 revealed that 10-12 M ET-1-(1---31) did not evoke Ca2+ influx into the cells, whereas ET-1-(1---31) at 10-7 M caused marked Ca2+ influx, as did ET-1 and Big ET-1. Moreover, the 10-7 M ET-1-(1---31)-induced Ca2+ influx was inhibited by nifedipine, a typical voltage-dependent Ca2+ channel blocker. Taken together, these data suggest that ET-1-(1---31) at low concentrations (pM range) causes [Ca2+]i increase via mobilization from intracellular stores, whereas marked Ca2+ influx occurs at relatively high concentrations (nM range), probably through voltage-dependent Ca2+ channels. These findings are consistent with the concept that release of Ca2+ from intracellular stores is a necessary prerequisite for the operation of plasma membrane Ca2+ channels (23). Considering the findings that the tissue or blood concentrations of ET-1 are in the picomolar range (5, 25), it is reasonable to speculate that the physiological activity of ET-1-(1---31) is attributable to Ca2+ mobilization from intracellular stores rather than the influx of Ca2+ from the extracellular space. Our results also show that ET-1 and Big ET-1 mediate their responses at picomolar concentrations via an intracellular mechanism and at nanomolar concentrations via Ca2+ influx at least in cultured human coronary artery smooth muscle cells.

In summary, ET-1-(1---31) increased [Ca2+]i in a concentration-dependent manner (10-14-10-10 M) in cultured human coronary artery smooth muscle cells. The ET-1-(1---31)-induced [Ca2+]i increase at relatively low concentrations (pM range) may be attributable to the release of Ca2+ from IP3-sensitive intracellular stores, whereas Ca2+ influx into the cells evoked by high concentration (nM range) of ET-1-(1---31) may occur through voltage-dependent Ca2+ channels. We conclude that the physiological activity of ET-1-(1---31) may be attributable to Ca2+ mobilization from intracellular stores rather than influx of Ca2+ from the extracellular space.


    ACKNOWLEDGEMENTS

This work was supported in part by Grants-in-Aid for Scientific Research (no. 10770040 and no. 10670085) from the Ministry of Education, Science, Sports, and Culture, Japan. We are grateful to Keiko Tachibana for secretarial assistance.


    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: D. Inui, Dept. of Pharmacology, The Univ. of Tokushima School of Medicine, 3-18-15 Kuramoto, Tokushima 770-8503, Japan (E-mail: yakuri{at}basic.med.tokushima-u.ac.jp).

Received 14 July 1998; accepted in final form 3 March 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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