AJP - Heart Calcium Transients and Cell-Sarcomere
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Am J Physiol Heart Circ Physiol 289: H1551-H1559, 2005; doi:10.1152/ajpheart.00131.2005
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Endothelin-1 increases intracellular Ca2+ in rabbit pulmonary artery smooth muscle cells through phospholipase C

Eun A. Ko,1 Won Sun Park,1 Jae-Hong Ko,1 Jin Han,2 Nari Kim,2 and Yung E. Earm1

1Department of Physiology and National Research Laboratory for Cellular Signalling, Seoul National University College of Medicine, Seoul; and 2Mitochondrial Signaling Laboratory, Department of Physiology and Biophysics, College of Medicine, Biohealth Products Research Center, Cardiovascular and Metabolic Disease Center, Inje University, Busan, Korea

Submitted 9 February 2005 ; accepted in final form 26 May 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
In freshly isolated rabbit pulmonary artery smooth muscle cells, endothelin (ET)-1 induced a transient increase in intracellular Ca2+ concentration ([Ca2+]i) followed by a return to the initial [Ca2+]i. This response was not abolished by the voltage-dependent Ca2+ channel blocker nicardipine or removal of Ca2+ from the bath solution but was inhibited by ryanodine and thapsigargin. This finding suggested that the increase in [Ca2+]i induced by ET-1 was attributable to release of Ca2+ from ryanodine- and inositol 1,4,5-trisphosphate-sensitive intracellular Ca2+ stores. The transient increase in [Ca2+]i induced by ET-1 was also inhibited by pretreatment with antagonists of ET type A and B (ETA and ETB) receptors (BQ-123 and BQ-788, respectively). Furthermore, the ETB receptor agonist IRL-1620 induced an increase in [Ca2+]i that was followed by a sustained increase in [Ca2+]i; the sustained increase in [Ca2+]i was blocked by nicardipine. Using the nystatin-perforated patch-clamp technique, we found that IRL-1620 caused an increase in Ca2+ current that was inhibited by addition of ET-1. ET-1 did not inhibit Ca2+ current when cells were pretreated with BQ-123. These results suggested that when both receptor types are activated, the opposing responses lead to abolition of the sustained [Ca2+]i increases induced by ETB receptor activation. Western blot analysis confirmed expression of ETA and ETB receptors. Finally, U-73122 inhibited the ET-1-induced [Ca2+]i increase, indicating that phospholipase C was involved in modulation of the ET-1-induced [Ca2+]i increase in rabbit pulmonary artery smooth muscle cells.

endothelin receptors; voltage-dependent Ca2+ channel

ENDOTHELIN (ET)-1, the most potent vasoconstrictor described to date, is a 21-amino acid peptide secreted by the vascular endothelium (44). After release from the endothelium, ET-1 induces smooth muscle contraction by binding to ET receptor type A or B (ETA or ETB) receptors. ETA receptors are present only on smooth muscle cells, whereas ETB receptors are located on endothelial and smooth muscle cells (35). Studies of the roles of the two receptor subtypes in mediating the effects of ET-1 have produced highly variable results that range from exclusive mediation by one of the receptor subtypes to an equivalent contribution by both types (4, 5, 22, 29, 31, 33, 45). The ratio and coupling efficacy of ETA and ETB receptors to the contractile apparatus of muscle cells may vary across species, vascular regions, and vessel size, as well as with the concentration of ET-1 (1, 6, 13, 15). Fukuroda et al. (6) reported that ETA receptors were dominant in the human pulmonary artery, whereas ETB receptors were dominant in the rabbit pulmonary artery. Because both receptor types are coupled G proteins, they activate several signaling pathways, including phospholipase C (PLC), resulting in generation of inositol 1,4,5-trisphosphate (IP3) and 1,2-diacylglycerol (DAG) (2, 23, 24). These molecules in turn cause an increase in the intracellular concentration of Ca2+ ([Ca2+]i), which activates protein kinase C (PKC) and leads to the contraction of smooth muscle (35, 42). However, the cellular mechanisms by which the ET-1 receptors induce an increase in [Ca2+]i in smooth muscle cells remain unclear. ET-1 could increase [Ca2+]i by stimulating Ca2+ influx through the plasmalemma (10, 45) and/or by causing Ca2+ to be released from internal Ca2+ stores (30, 41). The present study was designed to investigate 1) the source of the Ca2+ that is mobilized by ET-1, 2) the ET-1 receptor subtype(s) that mediates the effects of ET-1, and 3) the intracellular mechanisms that underlie the response of smooth muscle cells to ET-1.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell isolation. The procedure was carried out in accordance with the guidelines of the European Community on the ethical use of animals, and all experimental procedures were reviewed and approved by the Committee for Animal Experiments of the Seoul National University College of Medicine. New Zealand White rabbits (2–2.5 kg) of either gender were anesthetized with pentobarbital sodium (50 mg/kg). The lungs were removed immediately and immersed in normal Tyrode solution containing 143 mM NaCl, 5.4 mM KCl, 0.33 mM NaH2PO4, 1.8 mM CaCl2, 1.2 mM MgCl2, 5 mM HEPES, and 16.6 mM glucose (pH adjusted to 7.4 with NaOH). Small pulmonary arteries (<200 µm OD), derived after the third or fourth branching points of the intralobar pulmonary arteries in the lower lobe on either side of the lung, were isolated by dissection under a dissecting microscope. The arteries were incubated at 37°C in the first digestion medium (Ca2+-free Tyrode solution containing 1.0 mg/ml papain, 1.0 mg/ml bovine serum albumin, and 1.0 mg/ml dithiothreitol) for 20 min and in the second digestion medium (Ca2+-free Tyrode solution containing 1.5 mg/ml collagenase, 1.0 mg/ml bovine serum albumin, and 1.0 mg/ml dithiothreitol) for 25 min. The samples were rinsed with Ca2+-free normal Tyrode solution to release the enzymes, and the released cells were detached by gentle agitation with a fire-polished glass pipette. The isolated cells were suspended in a storage medium (70 mM KOH, 50 mM L-glutamate, 55 mM KCl, 20 mM taurine, 20 mM KH2PO4, 3 mM MgCl2, 20 mM glucose, 10 mM HEPES, and 0.5 mM EGTA) at 4°C and used within 12 h. Smooth muscle cells were identified morphologically and functionally by their ability to respond to depolarization with 100 mM KCl by contracting and increasing [Ca2+]i. Cells that did not exhibit a Ca2+ response to K+ depolarization were not used.

Measurement of intracellular Ca2+. Intracellular Ca2+ was measured using the membrane-permeant (acetoxymethyl ester) form of the Ca2+-sensitive fluorescent dye indo 1 (indo 1-AM). The cells were loaded by incubation in 3 µM indo 1-AM for 30 min at 37°C, and the extracellular indo 1-AM was then rinsed off with normal Tyrode solution. Monochromatic excitation light (355 nm) was delivered to the cell using a filter wheel (Life Science Resources, Cambridge, UK) via a liquid light guide and an oil-immersion objective lens (x40, NA 1.3; Nikon). The light emitted through an aperture slightly larger than the cell was measured simultaneously at 405 and 490 nm, and Ca2+ concentration was estimated from the ratio of the fluorescence signals (405/490) obtained from the two photomultipliers (Life Science Resources). [Ca2+]i was calculated using the equation described by Grynkiewicz et al. (12)

where R is the ratio of the fluorescence signals (405/490), Rmin is 405/490 in Ca2+-free medium containing 10 mM EGTA (ratio = 0.8), Rmax is 405/490 in the presence of saturating Ca2+ (ratio = 7.5), and {beta} is the ratio of the 490-nm fluorescence signal measured in a Ca2+-free solution to that measured in a Ca2+-replete solution (ratio = 8.2). The Ca2+ dissociation constant (Kd) was determined to be 250 nM at 37°C for this dye and optical system. All experiments were carried out at room temperature (22–25°C).

Perforated-patch recordings. Membrane currents were recorded using a patch-clamp amplifier (Axopatch 1C, Axon Instrument, Union, CA). Pulse protocols and data acquisition were performed by a digital interface (Digidata 1200, Axon Instrument) coupled to an IBM-compatible microcomputer. The current signals were filtered at 0.5–1 kHz, and the sampling rate was 1–3 kHz. For the perforated-patch recordings of the voltage-dependent Ca2+ currents, the cells were bathed in a solution containing 120 mM NaCl, 2 mM CaCl2, 5 mM CsCl, 20 mM TEA-Cl, 0.5 mM MgCl2, 10 mM HEPES, and 10 mM glucose (pH adjusted to 7.4 with NaOH). The patch pipettes were filled with a solution containing 130 mM CsCl, 10 mM HEPES, 10 mM EGTA, and 5 mM Mg-ATP (pH adjusted to 7.2 with CsOH). Nystatin was added to a fresh aliquot of the pipette solution every 2 h to give a final concentration of 200 µg/ml. Inward currents through voltage-gated Ca2+ channels were recorded by application of a depolarizing pulse to 0 mV for 300 ms from the holding potential of –80 mV every 10 s. Membrane capacitance was determined using 10-mV voltage-clamp steps from a holding potential of –60 mV, and the current amplitudes were normalized using the membrane capacitance. The average cell capacitance was 14.9 ± 1.59 (SE) pF (n = 24).

Western blot analysis. Sections of the rabbit pulmonary arteries were homogenized in a hand-held microtissue grinder (Pyrex, Corning Life Sciences, Acton, MA) in two volumes of ice-cold storage buffer (100 mM KPO4, 1 mM EDTA, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, and 30% glycerol, pH 7.25). The homogenates were centrifuged at 3,500 g for 15 min at 4°C. The supernatants were colleted and stored at –80°C until use in Western blot analysis. Aliquots of pulmonary artery homogenates containing 20 µg of protein were separated by electrophoresis on 10% SDS-polyacrylamide gels. The gels were transferred to Immobilon-P membranes (Millipore), which were blocked overnight in Tris-buffered saline (20 mM Tris and 150 mM NaCl, pH 8.0) containing 5% nonfat dry milk and then probed with antiserum to {beta}-tubulin, ETA receptor, or ETB receptor at 1:200 dilution for 1 h at room temperature. The membranes were then incubated with horseradish peroxidase-conjugated goat anti-mouse (for {beta}-tubulin) or mouse anti-goat (for ETA and ETB receptors) antibody at 1:1,000 dilution for 1 h at room temperature. The bands were visualized using an enhanced chemiluminescence Western blotting detection kit (Amersham Biosciences, Piscataway, NJ).

Chemicals and drugs. Indo 1-AM was purchased from Molecular Probes (Eugene, OR), collagenase from Wako Pure Chemical Industries (Osaka, Japan), and antisera to ETA and ETB and mouse anti-goat antibody from Santa Cruz Biotechnology (Santa Cruz, CA). All other chemicals were obtained from Sigma Chemical (St. Louis, MO).

Statistics. Values are means ± SE. Differences were examined for significance using Student's t-test. P < 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Transient Ca2+ response to ET-1 does not involve extracellular Ca2+. Exposure of rabbit pulmonary artery smooth muscle cells to ET-1 caused a rapid rise in [Ca2+]i followed by a return to baseline Ca2+ levels. The magnitude of this transient peak was concentration dependent, with maximum [Ca2+]i of 28.6 ± 2.8 nM (n = 8), 46.9 ± 2.5 nM (n = 11), and 82.4 ± 3.7 nM (n = 15) at 0.25, 0.5, and 5 nM ET-1, respectively (Fig. 1, A and B). [Ca2+]i increased to 176.1 ± 5.6 nM (n = 18) in cells that were depolarized with 100 mM KCl (Fig. 1D), indicating that the cells were responsive and that the sarcoplasmic reticulum was loaded with Ca2+ (35, 38). After they were washed for 15 min, the cells were exposed to ET-1. To determine whether the ET-1-induced increase in [Ca2+]i required Ca2+ influx through voltage-dependent channels, 5 nM ET-1 was applied 10 min after the start of exposure to 5 µM nicardipine, an L-type Ca2+ channel antagonist. Nicardipine had no effect on the ET-1-induced increase in [Ca2+]i (n = 6; Fig. 1C). To further evaluate the role of extracellular Ca2+ influx, 5 nM ET-1 was applied to cells that were superfused with Ca2+-free buffer containing 2 mM EGTA for 7 min before stimulation with ET-1. Removal of extracellular Ca2+ with EGTA did not affect the transient [Ca2+]i increase induced by ET-1 (n = 7; Fig. 1D). Therefore, we concluded that, in pulmonary artery smooth muscle cells, ET-1-induced [Ca2+]i increases are not mediated by influx of extracellular Ca2+.



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  		Fig. 1. Endothelin (ET)-1-induced increases in intracellular Ca2+ concentration ([Ca2+]i) are not mediated by extracellular Ca2+. A: changes in [Ca2+]i induced by 0.25, 0.5, and 5 nM ET-1. B: average maximal [Ca2+]i in response to ET-1. C and D: effect of Ca2+ channel blockade with 5 µM nicardipine (C) and removal of extracellular Ca2+ with EGTA (D) on increases in [Ca2+]i induced by 5 nM ET-1. Vertical bars indicate 20-min gap in recording. Traces are representative of data from 7–15 cells. *P < 0.01; #P < 0.001 vs. baseline.

 
ET-1 induces Ca2+ release from intracellular stores. Given that the removal of extracellular Ca2+ did not abolish ET-1-induced increases in [Ca2+]i, the contribution of the release of Ca2+ from intracellular stores was examined. To determine which intracellular Ca2+ stores were involved in the ET-1-induced increase in [Ca2+]i, we used ryanodine to block the ryanodine-sensitive intracellular Ca2+ stores (20) or thapsigargin (38) to block the endoplasmic reticulum Ca2+-ATPase pump. The ET-1-induced [Ca2+]i increase was attenuated in the presence of 10 µM ryanodine (Fig. 2A). In addition, pretreatment of the cells with 10 µM thapsigargin completely abolished the ET-1-induced [Ca2+]i increase (Fig. 2B). These results (summarized in Fig. 2C) suggested that intracellular ryanodine- and thapsigargin-sensitive stores of Ca2+ were mobilized by ET-1 stimulation.



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  		Fig. 2. ET-1 evokes increases in [Ca2+]i through mobilization of intracellular Ca2+ stores. A and B: changes in [Ca2+]i after addition of 5 nM ET-1 to cells after pretreatment with 10 µM ryanodine (A) and 1 µM thapsigargin (B) for 10 min. Vertical bars indicate 20-min gap in recording. Traces are representative of 5 experiments. C: average maximal [Ca2+]i in response to ET-1 in the presence of ryanodine or thapsigargin. *P < 0.01; #P < 0.001 vs. ET-1.

 
ETA and ETB receptors mediate the Ca2+ response to ET-1. We investigated the involvement of the two ET-1 receptor subtypes in the ET-1-induced increase in [Ca2+]i by using BQ-123 and BQ-788 to antagonize ETA and ETB receptors, respectively (17, 19), as well as IRL-1620, a specific agonist of ETB receptors (37). The ET-1-induced [Ca2+]i increase was partially blocked in the presence of 1 µM BQ-123 (n = 5; Fig. 3A), which suggested that the ETA receptor partially mediated the Ca2+ response to ET-1. The ET-1-induced [Ca2+]i increase was completely inhibited by 1 µM BQ-788, although 100 mM KCl could still evoke an increase in [Ca2+]i (n = 8; Fig. 3B), suggesting that the ET-1-induced [Ca2+]i increase occurred mainly via ETB receptors. Subsequent application of 5 nM ET-1 failed to induce an increase in [Ca2+]i (n = 11; Fig. 3C). In contrast, the effect of 10 nM IRL-1620 on [Ca2+]i was reversible after a 20-min wash (n = 12; Fig. 3D), indicating that two different types of receptors were present in the pulmonary artery smooth muscle cells. To demonstrate that ET-1 could induce Ca2+ influx through L-type voltage-dependent Ca2+ channels by ETB receptor activation in the absence of ETA receptor activity, cells pretreated with 1 µM BQ-123 were exposed to nicardipine on the sustained component of ET-1-induced Ca2+ increases. The sustained increase in [Ca2+]i by ET-1 was completely abolished by treatment with 1 µM nicardipine (n = 5; Fig. 3E), indicating that the sustained component of the Ca2+ response to ET-1 in the absence of ETA receptor activity was mediated by the influx of extracellular Ca2+ via voltage-dependent Ca2+ channels by ETB receptor activation. Perfusion of 10 nM IRL-1620 into the cells caused [Ca2+]i to increase to 108.8 ± 4.9 nM (n = 11). IRL-1620 elicited an initial transient peak in [Ca2+]i followed by a sustained elevation of [Ca2+]i. The sustained increase in [Ca2+]i was abolished by 1 µM nicardipine (n = 5; Fig. 3F), indicating that the sustained component of the Ca2+ response to IRL-1620 was attributable to the influx of extracellular Ca2+ via voltage-dependent Ca2+ channels. However, the sustained component was not observed in cells that were exposed to ET-1. These findings suggested that there are two separate mechanisms for Ca2+ release, each of which is selectively activated by ETA or ETB receptors and ET-1-evoked [Ca2+]i increases mainly through ETB receptor activation with partial ETA receptor activation.



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  		Fig. 3. ET-1-induced [Ca2+]i increases are mediated by ET type A and B (ETA and ETB) receptors. A and B: changes in [Ca2+]i induced by ET-1 after pretreatment with the ETA receptor blocker BQ-123 (1 µM; A) or the ETB receptor blocker BQ-788 (1 µM; B). C and D: changes in [Ca2+]i induced by repeated addition of 5 nM ET-1 (C) or the ETB receptor agonist IRL-1620 (10 nM; D). E: sustained component of the [Ca2+]i increase induced by 5 nM ET-1 was inhibited by 1 µM nicardipine in cells pretreated with 1 µM BQ-123. F: sustained component of the [Ca2+]i increase induced by 10 nM IRL-1620 was inhibited by 1 µM nicardipine. Vertical bars indicate 20-min gap in recording. Traces are representative of 5–12 experiments. G: summarized data normalized to initial peak [Ca2+]i on the first stimulation by ET-1 and IRL-1620. *P < 0.01; #P < 0.001 vs. ET-1.

 
Interaction of ETA and ETB receptors determines the Ca2+ response to ET-1. The lack of a sustained increase in [Ca2+]i in response to ET-1 might be caused by ETA receptor activation, which causes inhibition of the influx of extracellular Ca2+ through Ca2+ channels activated by the ETB receptor. Using a perforated patch-clamp technique, we examined the effect of ET-1 on changes in Ca2+ current (ICa) evoked by IRL-1620. IRL-1620 (100 nM) increased ICa by 65.4 ± 2.8% (n = 18; Fig. 4C) compared with the control value. This effect was completely inhibited by 1 µM BQ-788 (n = 8; Fig. 4A) and could be reversed on washout of IRL-1620 (data not shown), which suggested that IRL-1620 increased ICa mainly via activation of ETB receptors. Addition of 30 nM ET-1 alone had no significant effect on ICa (n = 12; Fig. 4B), but the increase in ICa induced by 100 nM IRL-1620 was markedly attenuated by 30 nM ET-1 (n = 14; Fig. 4, D and E). Addition of 1 µM BQ-123 did not affect basal ICa (data not shown); however, in cells pretreated with 1 µM BQ-123, ICa activated by 100 nM IRL-1620 was not inhibited by 30 nM ET-1 (n = 6; Fig. 4, F and G), which suggested that the inhibition of ICa caused by ET-1 in the absence of BQ-123 occurred via ETA receptor activation, rather than a nonspecific effect. These observations indicated that the absence of a sustained increase in [Ca2+]i in response to ET-1 may be the result of activation of ETA receptors, which masks the Ca2+ influx through Ca2+ channels activated by ETB receptors. To exclude the possibility that IRL-1620 activates ICa directly, rather than through the ETB receptor, we examined the effect of ET-1 on ICa in the presence of the ETA antagonist BQ-123. ET-1 increased ICa by 45.3 ± 4.3% (n = 5; Fig. 4, H and I) in the absence of ETA activity, which suggested that ETB receptor activation could increase ICa.



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  		Fig. 4. ET-1 inhibits changes in Ca2+ current (ICa) evoked by IRL-1620. Original current traces recorded from a holding potential of –80 to 0 mV show changes in ICa evoked by application of 100 nM IRL-1620 (A) and 30 nM ET-1 (B). C: current density of ICa evoked by IRL-1620. *P < 0.01 vs. baseline. D: changes in ICa induced by 30 nM ET-1 in the presence of 100 nM IRL-1620. E: current density-time relations of ICa. F: changes in ICa evoked by application of 30 nM ET-1 in the presence of 100 nM IRL-1620 after pretreatment with 1 µM BQ-123. G: current density-time relations of ICa. H: increases in ICa induced by 30 nM ET-1 in the presence of BQ-123. I: current density-time relations of ICa. Traces are representative of 5–14 independent experiments.

 
ETA and ETB receptors are expressed in pulmonary artery smooth muscle cells. To confirm that rabbit pulmonary artery smooth muscle cells expressed ETA and ETB receptors, we performed an immunoblot analysis of pulmonary membrane proteins using anti-ETA and anti-ETB affinity-purified antisera. Each antiserum recognized protein bands of the expected molecular mass, indicating that both receptor subtypes are expressed in rabbit pulmonary artery smooth muscle cells (Fig. 5). These results support our hypothesis that ET-1 interacts with ETA and ETB receptors to increase [Ca2+]i.



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  		Fig. 5. Immunoblot analysis of expression of ETA and ETB receptors in rabbit endothelium-denuded pulmonary arteries. Western blotting was performed using anti-ETA and anti-ETB receptor antibodies. 45-kDa immunoreactive band corresponds to the known molecular size of the ETA receptor; 50-kDa immunoreactive band corresponds to the known molecular size of the ETB receptor.

 
Ca2+ response to ET-1 involves PLC. The transient [Ca2+]i increase brought about by stimulation of the ET-1 receptors may be produced by activation of PLC (2, 23, 24). We used U-73122, a membrane-permeant inhibitor of PLC, to test whether activation of PLC linked to ET-1 receptors could increase [Ca2+]i in rabbit pulmonary artery smooth muscle cells. Incubation of cells with 3 µM U-73122 caused a complete inhibition of the ET-1-induced [Ca2+]i increase (n = 4; Fig. 6A), which suggested that the ET-1-induced increase in [Ca2+]i involved activation of PLC. In addition, incubation of cells with U-73122 caused a complete inhibition of the IRL-1620-induced [Ca2+]i increase (n = 4; Fig. 6C). Also, treatment with U-73122 completely inhibited the ICa activated by IRL-1620 (n = 5; Fig. 6E). Basal ICa was not affected by treatment with U-73122 (data not shown), which suggested that effects on [Ca2+]i and ICa induced by ETB receptor activation involved PLC. Stimulation of PLC in response to the occupation of ET-1 receptors is associated with generation of IP3 and DAG; the latter activates multiple effectors via PKC phosphorylation, including ryanodine receptors and L-type Ca2+ channels (11, 43).



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  		Fig. 6. ET-1-induced [Ca2+]i increases are inhibited by phospholipase C blockers. A: changes in [Ca2+]i induced by ET-1 after pretreatment with the phospholipase C inhibitor U-73122 (3 µM). B: summarized data from A. #P < 0.001 vs. ET-1. C: inhibition of increases in [Ca2+]i induced by 10 nM IRL-1620 after treatment with 3 µM U-73122. D: summarized data from C. #P < 0.001 vs. IRL-1620. Vertical bars indicate 20-min gap in recording. E: inhibition of ICa activated by 100 nM IRL-1620 after treatment with 3 µM U-73122. F: current density-time relations of ICa. Traces are representative of 4–5 experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
We characterized the cellular mechanisms that underlie ET-1-induced increases in [Ca2+]i in rabbit pulmonary artery smooth muscle cells. ET-1 induced a transient increase in [Ca2+]i that was followed by a return to baseline levels. Our results revealed patterns of changes in [Ca2+]i that are distinct from those reported elsewhere. For example, ET-1 was reported to induce a large transient [Ca2+]i increase followed by a sustained plateau that remained elevated above baseline (8, 34), and other studies reported oscillations in [Ca2+]i in smooth muscle cells in response to ET-1 (3, 16). This discrepancy is most likely attributable to differences in the wide variety of models used to study Ca2+ regulation, which include freshly isolated cells, cultured cells, and different cell types. Godfraind (9) suggested that ETA receptors mediate constriction in smaller, preresistance arteries, whereas more than one ET receptor is involved in the larger segments. In contrast, Adner and colleagues (1) indicated a dominance of ETB receptors in the human peripheral lung. Therefore, participation of ET-1 receptor subtypes might differ according to artery diameter, which is attributed to the different pattern of [Ca2+]i response to ET-1. In our study, we clarified the effect of ET-1-induced [Ca2+]i increases on <200-µm third- to fourth-order pulmonary resistance arteries. We found that ETA and ETB receptors coexist in this preparation, as shown in Western blot analysis, and the ET-1-induced [Ca2+]i increases were mediated mainly through ETB receptors.

Our results clearly showed that the ET-1-induced [Ca2+]i response in rabbit pulmonary artery smooth muscle cells was the result of the release of [Ca2+]i from ryanodine- and IP3-sensitive intracellular stores, inasmuch as the response was not altered by nicardipine or removal of Ca2+ from the bath solution but was inhibited by pretreatment of the cells with ryanodine or thapsigargin. The mechanisms that mediate the effects of ET-1 on smooth muscle cells are unclear. An earlier study using patch-clamp techniques revealed that voltage-gated Ca2+ channels were activated by ET-1; removal of extracellular Ca2+ and pretreatment of cells with verapamil reduced the ET-1-induced transient increase in [Ca2+]i in vascular smooth muscle (10, 18, 34). However, other evidence suggested that inhibitors of L-type voltage-gated Ca2+ channels did not inhibit the ET-1-induced [Ca2+]i increase or the contraction of vascular smooth muscle (25, 32).

In the present study, BQ-788 completely inhibited the ET-1-induced [Ca2+]i increase (Fig. 3B), which suggested that the activation of ETB receptors is necessary for the ET-1-induced increase in [Ca2+]i. However, the patterns of changes in [Ca2+]i induced by IRL-1620 from those induced by ET-1. IRL-1620 induced a sustained, rather than a transient, [Ca2+]i increase. The sustained component resulted from Ca2+ influx through voltage-dependent Ca2+ channels, because it was abolished when cells were treated with nicardipine (Fig. 3E). Using the nystatin-perforated patch-clamp technique, we found that the activation of ETB receptors increased ICa and that ICa was inhibited by the activation of ETA receptors. Our data suggested that the two subtypes of ET-1 receptors produce increases in [Ca2+]i that are the net result of a complex interaction between the regulation of Ca2+ channels by each receptor subtype. The opposing effects of ETA and ETB receptors on [Ca2+]i lead to the elimination of a sustained [Ca2+]i increase when both receptors are activated.

In this study, we found that IRL-1620 had no effect on ICa measured using the whole cell patch-clamp technique (data not shown). Such differences in experimental techniques have contributed to the apparent discrepancies in the reported results. The perforated patch-clamp recording allows ICa to be studied without the movement of large intracellular molecules and structures that occurs with the rupture of the cell membranes required in the whole cell patch-clamp technique (14, 21). Our results indicated that ETA and ETB receptors cooperate positively to mediate the transient component of the ET-1-induced [Ca2+]i increase and cooperate negatively to lead to the elimination of the sustained [Ca2+]i increases induced by ETB receptor activation in rabbit pulmonary artery smooth muscle cells. In agreement with our results, it has been reported that both ET receptors can contribute to ET-1-induced contraction; for example, in pulmonary arteries, both receptors coexist and, thus, could cooperate to mediate contraction (6, 7, 22).

In general, the ET-1-induced increase in [Ca2+]i involves different Ca2+-mobilizing mechanisms, including release of Ca2+ from intracellular stores via a PLC-mediated activation of IP3 receptors, activation of L-type Ca2+ channels, and Ca2+-activated Cl– currents (27, 28, 34, 40, 43). In our study, the ET-1-induced increases in [Ca2+]i were not inhibited by the Ca2+ channel blocker nicardipine, and ET-1 did not affect ICa. Therefore, we believe that Ca2+ release from intracellular stores is the main mechanism by which ET-1 elicits an increase in [Ca2+]i. Furthermore, ET-1-induced [Ca2+]i increases were completely abolished by the PLC blocker U-73122, which suggested that the ET-1-induced Ca2+ responses involve activation of PLC. Previous reports have shown that PLC plays a crucial role in ET-1-induced [Ca2+]i increases in cultured ciliary muscle and bronchial smooth muscle cells (26, 27). However, further investigations should explore whether activation of the PLC pathway results in the generation of two second messengers, IP3 and DAG, which are involved in intracellular Ca2+ release and PKC activation, respectively.

In conclusion, we have demonstrated that ET-1 induces the release of Ca2+ from intracellular stores in rabbit pulmonary artery smooth muscle cells via activation of ETA and ETB receptors. In addition, ET-1 was functionally coupled to a rise in [Ca2+]i through activation of PLC.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by the Brain Korea 21 project for Human Life Sciences of the Korean Ministry of Education and a National Research Laboratory Grant from the Korean Ministry of Science and Technology.


   FOOTNOTES
 

Address for reprint requests and other correspondence: Y. E. Earm, Dept. of Physiology, College of Medicine, Seoul National Univ., Seoul 110-799, Korea (E-mail: earmye{at}sun.ac.kr)

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   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Adner M, Cardell LO, Sjoberg T, Ottosson A, and Edvinsson L. Contractile endothelin-B (ETB) receptors in human small bronchi. Eur Respir J 9: 351–355, 1996.[Abstract]
  2. Araki S, Kawahara Y, Kariya K, Sunako M, Fukuzaki H, Takai Y. Stimulation of phospholipase C-mediated hydrolysis of phosphoinositides by endothelin in cultured rabbit aortic smooth muscle cells. Biochem Biophys Res Commun 159: 1072–1079, 1989.[CrossRef][ISI][Medline]
  3. Bakhramov A, Hartley SA, Salter KJ, and Kozlowski RZ. Contractile agonists preferentially activate Cl– over K+ currents in arterial myocytes. Biochem Biophys Res Commun 227: 168–175, 1996.[CrossRef][ISI][Medline]
  4. Berthiaume N, Claing A, Yano M, and D'Orleans-Juste P. Characterization of receptors for endothelins in the guinea pig mesenteric vasculature. J Cardiovasc Pharmacol 26: S317–S319, 1995.[Medline]
  5. Evans AM, Cobban HJ, and Nixon GF. ETA receptors are the primary mediators of myofilament calcium sensitization induced by ET-1 in rat pulmonary artery smooth muscle: a tyrosine kinase independent pathway. Br J Pharmacol 127: 153–160, 1999.[CrossRef][ISI][Medline]
  6. Fukuroda T, Kobayashi M, Ozaki S, Yano M, Miyauchi T, Onizuka M, Sugishita Y, Goto K, and Nishikibe M. Endothelin receptor subtypes in human versus rabbit pulmonary arteries. J Appl Physiol 76: 1976–1982, 1994.[Abstract/Free Full Text]
  7. Fukuroda T, Ozaki S, Ihara M, Ishikawa K, Yano M, and Nishikibe M. Synergistic inhibition by BQ-123 and BQ-788 of endothelin-1-induced contractions of the rabbit pulmonary artery. Br J Pharmacol 113: 336–338, 1994.[ISI][Medline]
  8. Gardner JP, Tokudome G, Tomonari H, Maher E, Hollander D, and Aviv A. Endothelin-induced calcium responses in human vascular smooth muscle cells. Am J Physiol Cell Physiol 262: C148–C155, 1992.[Abstract/Free Full Text]
  9. Godfraind T. Evidence for heterogeneity of endothelin receptor distribution in human coronary artery. Br J Pharmacol 110: 1201–1205, 1993.[ISI][Medline]
  10. Goto K, Kasuya Y, Matsuki N, Takuwa Y, Kurihara H, Ishikawa T, Kimura S, Yanagisawa M, and Masaki T. Endothelin activates the dihydropyridine-sensitive, voltage-dependent Ca2+ channel in vascular smooth muscle. Proc Natl Acad Sci USA 86: 3915–3918, 1989.[Abstract/Free Full Text]
  11. Griendling KK, Tsuda T, and Alexander RW. Endothelin stimulates diacylglycerol accumulation and activates protein kinase C in cultured vascular smooth muscle cells. J Biol Chem 264: 8237–8240, 1989.[Abstract/Free Full Text]
  12. Grynkiewicz G, Poenie M, and Tsien RY. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 260: 3440–3450, 1985.[Abstract/Free Full Text]
  13. Hay DW, Luttmann MA, Beck G, and Ohlstein EH. Comparison of endothelin B (ETB) receptors in rabbit isolated pulmonary artery and bronchus. Br J Pharmacol 118: 1209–1217, 1996.[ISI][Medline]
  14. He JQ, Pi Y, Walker JW, and Kamp TJ. Endothelin-1 and photoreleased diacylglycerol increase L-type Ca2+ current by activation of protein kinase C in rat ventricular myocytes. J Physiol 524: 807–820, 2000.[Abstract/Free Full Text]
  15. Higashi T, Ishizaki T, Shigemori K, Nakai T, Miyabo S, Inui T, and Yamamura T. Pharmacological heterogeneity of constrictions mediated by endothelin receptors in rat pulmonary arteries. Am J Physiol Lung Cell Mol Physiol 272: L287–L293, 1997.[Abstract/Free Full Text]
  16. Hyvelin JM, Guibert C, Marthan R, and Savineau JP. Cellular mechanisms and role of endothelin-1-induced calcium oscillations in pulmonary arterial myocytes. Am J Physiol Lung Cell Mol Physiol 275: L269–L282, 1998.[Abstract/Free Full Text]
  17. Ihara M, Ishikawa K, Fukuroda T, Saeki T, Funabashi K, Fukami T, Suda H, and Yano M. In vitro biological profile of a highly potent novel endothelin (ET) antagonist BQ-123 selective for the ETA receptor. J Cardiovasc Pharmacol 20: S11–S14, 1992.
  18. Inoue Y, Oike M, Nakao K, Kitamura K, and Kuriyama H. Endothelin augments unitary calcium channel currents on the smooth muscle cell membrane of guinea-pig portal vein. J Physiol 423: 171–191, 1990.[Abstract/Free Full Text]
  19. Ishikawa K, Ihara M, Noguchi K, Mase T, Mino N, Saeki T, Fukuroda T, Fukami T, Ozaki S, and Nagase T. Biochemical and pharmacological profile of a potent and selective endothelin B-receptor antagonist, BQ-788. Proc Natl Acad Sci USA 91: 4892–4896, 1994.[Abstract/Free Full Text]
  20. Ito K, Takakura S, Sato K, and Sutko JL. Ryanodine inhibits the release of calcium from intracellular stores in guinea pig aortic smooth muscle. Circ Res 58: 730–734, 1986.[Abstract/Free Full Text]
  21. Korn SJ, Marty A, Connor JA, and Horn R. Perforated patch recording. In: Methods in Neurosciences. New York: Academic, 1991, vol. 4, p. 364–373.
  22. LaDouceur DM, Flynn MA, Keiser JA, Reynolds E, and Haleen SJ. ETA and ETB receptors coexist on rabbit pulmonary artery vascular smooth muscle mediating contraction. Biochem Biophys Res Commun 196: 209–215, 1993.[CrossRef][ISI][Medline]
  23. Little PJ, Neylon CB, Tkachuk VA, and Bobik A. Endothelin-1 and endothelin-3 stimulate calcium mobilization by different mechanisms in vascular smooth muscle. Biochem Biophys Res Commun 183: 694–700, 1992.[CrossRef][ISI][Medline]
  24. Marsden PA, Danthuluri NR, Brenner BM, Ballermann BJ, and Brock TA. Endothelin action on vascular smooth muscle involves inositol trisphosphate and calcium mobilization. Biochem Biophys Res Commun 158: 86–93, 1989.[CrossRef][ISI][Medline]
  25. Masaki T, Kimura S, Yanagisawa M, and Goto K. Molecular and cellular mechanism of endothelin regulation. Implications for vascular function. Circulation 84: 1457–1468, 1991.[Free Full Text]
  26. Matsumoto S, Yorio T, Magnino PE, DeSantis L, and Pang IH. Endothelin-induced changes of second messengers in cultured human ciliary muscle cells. Invest Ophthalmol Vis Sci 37: 1058–1066, 1996.[Abstract/Free Full Text]
  27. Mattoli S, Soloperto M, Mezzetti M, and Fasoli A. Mechanisms of calcium mobilization and phosphoinositide hydrolysis in human bronchial smooth muscle cells by endothelin 1. Am J Respir Cell Mol Biol 5: 424–430, 1991.[Medline]
  28. Maxwell MJ, Goldie RG, and Henry PJ. Ca2+ signalling by endothelin receptors in rat and human cultured airway smooth muscle cells. Br J Pharmacol 125: 1768–1778, 1998.[CrossRef][ISI][Medline]
  29. Mickley EJ, Gray GA, and Webb DJ. Activation of endothelin ETA receptors masks the constrictor role of endothelin ETB receptors in rat isolated small mesenteric arteries. Br J Pharmacol 120: 1376–1382, 1997.[CrossRef][ISI][Medline]
  30. Mitsuhashi T, Morris RC Jr, and Ives HE. Endothelin-induced increases in vascular smooth muscle Ca2+ do not depend on dihydropyridine-sensitive Ca2+ channels. J Clin Invest 84: 635–639, 1989.[ISI][Medline]
  31. Nagase T, Aoki T, Oka T, Fukuchi Y, and Ouchi Y. ET-1-induced bronchoconstriction is mediated via ETB receptor in mice. J Appl Physiol 83: 46–51, 1997.[Abstract/Free Full Text]
  32. Satoh M, Yamamoto Y, and Takayanagi I. Characterization of endothelin receptor subtypes mediating Ca2+ mobilization and contractile response in rabbit iris dilator muscle. Br J Pharmacol 117: 1277–1285, 1996.[ISI][Medline]
  33. Seo B, Oemar BS, Siebenmann R, von Segesser L, and Luscher TF. Both ETA and ETB receptors mediate contraction to endothelin-1 in human blood vessels. Circulation 89: 1203–1208, 1994.[Abstract/Free Full Text]
  34. Shimoda LA, Sylvester JT, and Sham JS. Mobilization of intracellular Ca2+ by endothelin-1 in rat intrapulmonary arterial smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 278: L157–L164, 2000.[Abstract/Free Full Text]
  35. Simonson MS. Endothelins: multifunctional renal peptides. Physiol Rev 73: 375–411, 1993.[Free Full Text]
  36. Stehno-Bittel L, Laughlin MH, and Sturek M. Exercise training alters Ca2+ release from coronary smooth muscle sarcoplasmic reticulum. Am J Physiol Heart Circ Physiol 259: H643–H647, 1990.[Abstract/Free Full Text]
  37. Takai M, Umemura I, Yamasaki K, Watakabe T, Fujitani Y, Oda K, Urade Y, Inui T, Yamamura T, and Okada T. A potent and specific agonist, Suc-[Glu9,Ala11,15]-endothelin-1(8–21), IRL1620, for the ETB receptor. Biochem Biophys Res Commun 184: 953–959, 1992.[CrossRef][ISI][Medline]
  38. Thastrup O. Role of Ca2+-ATPases in regulation of cellular Ca2+ signalling, as studied with the selective microsomal Ca2+-ATPase inhibitor, thapsigargin. Agents Actions 29: 8–15, 1990.[CrossRef][ISI][Medline]
  39. Van Breemen C, Saida K, Yamamoto H, Hwang K, and Twort C. Vascular smooth muscle sarcoplasmic reticulum. Function and mechanisms of Ca2+ release. Ann NY Acad Sci 522: 60–73, 1988.[ISI][Medline]
  40. Van Renterghem C and Lazdunski M. Endothelin and vasopressin activate low conductance chloride channels in aortic smooth muscle cells. Pflügers Arch 425: 156–163, 1993.[CrossRef][ISI][Medline]
  41. Wagner-Mann C, Bowman L, and Sturek M. Primary action of endothelin on Ca2+ release in bovine coronary artery smooth muscle cells. Am J Physiol Cell Physiol 260: C763–C770, 1991.[Abstract/Free Full Text]
  42. Xuan YT, Wang OL, and Whorton AR. Regulation of endothelin-induced Ca2+ mobilization in smooth muscle cells by protein kinase C. Am J Physiol Cell Physiol 266: C1560–C1567, 1994.[Abstract/Free Full Text]
  43. Xuan YT, Whorton AR, and Watkins WD. Inhibition by nicardipine of endothelin-mediated inositol phosphate formation and Ca2+ mobilization in smooth muscle cell. Biochem Biophys Res Commun 160: 758–764, 1989.[CrossRef][ISI][Medline]
  44. Yanagisawa M, Kurihara H, Kimura S, Tomobe Y, Kobayashi M, Mitsui Y, Yazaki Y, Goto K, and Masaki T. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature 332: 411–415, 1988.[CrossRef][Medline]
  45. Yoshida M, Suzuki A, and Itoh T. Mechanisms of vasoconstriction induced by endothelin-1 in smooth muscle of rabbit mesenteric artery. J Physiol 477: 253–265, 1994.[Abstract/Free Full Text]



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