ET-1 activates Ca2+ sparks in PASMC: local Ca2+ signaling between inositol trisphosphate and ryanodine receptors

Wei-Min Zhang,1 Kay-Pong Yip,2 Mo-Jun Lin,1 Larissa A. Shimoda,1 Wen-Hong Li,3 and James S. K. Sham1

1Division of Pulmonary and Critical Care Medicine, Johns Hopkins School of Medicine, Baltimore, Maryland 21224; 2Department of Physiology and Biophysics, College of Medicine, University of South Florida, Tampa, Florida 33612; and 3Department of Cell Biology and Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas 75390

Submitted 11 March 2003 ; accepted in final form 5 May 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSIONS
 DISCLOSURES
 REFERENCES
 
Ca+ sparks originating from ryanodine receptors (RyRs) are known to cause membrane hyperpolarization and vasorelaxation in systemic arterial myocytes. By contrast, we have found that Ca2+ sparks of pulmonary arterial smooth muscle cells (PASMCs) are associated with membrane depolarization and activated by endothelin-1 (ET-1), a potent vasoconstrictor that mediates/modulates acute and chronic hypoxic pulmonary vasoconstriction. In this study, we characterized the effects of ET-1 on the physical properties of Ca2+ sparks and probed the signal transduction mechanism for spark activation in rat intralobar PASMCs. Application of ET-1 at 0.1-10 nM caused concentration-dependent increases in frequency, duration, and amplitude of Ca2+ sparks. The ET-1-induced increase in spark frequency was inhibited by BQ-123, an ETA-receptor antagonist; by U-73122, a PLC inhibitor; and by xestospongin C and 2-aminoethyl diphenylborate, antagonists of inositol trisphosphate (IP3) receptors (IP3Rs). However, it was unrelated to sarcoplasmic reticulum Ca2+ content, activation of L-type Ca2+ channels, PKC, or cADP ribose. Photorelease of caged-IP3 indicated that Ca2+ release from IP3R could cross-activate RyRs to generate Ca2+ sparks. Immunocytochemistry showed that the distributions of IP3Rs and RyRs were similar in PASMCs. Moreover, inhibition of Ca2+ sparks with ryanodine caused a significant rightward shift in the ET-1 concentration-tension relationship in pulmonary arteries. These results suggest that ET-1 activation of Ca2+ sparks is mediated via the ETA receptor-PLC-IP3 pathway and local Ca2+ cross-signaling between IP3Rs and RyRs; in addition, this novel signaling mechanism contributes significantly to the ET-1-induced vasoconstriction in pulmonary arteries.

endothelin; pulmonary arteries; photorelease; pulmonary artery smooth muscle cells; endothelin-1; calcium ion


ELEVATION OF INTRACELLULAR Ca2+, a ubiquitous signal for numerous cellular functions, is controlled by multiple pathways of transmembrane Ca2+ influx and intracellular Ca2+ release from inositol trisphosphate receptor (IP3R)- and ryanodine receptor (RyR)-gated Ca2+ stores. In arterial myocytes, the IP3R-gated Ca2+ store is thought to be the major source of agonist-induced Ca2+ release, responsible for Ca2+ waves and global intracellular Ca2+ concentration ([Ca2+]i) elevations for muscle contraction (21, 23, 30). By contrast, thorough studies in cerebral arteries showed that coordinated local Ca2+ releases from clusters of RyRs, or "Ca2+ sparks", function as the frequency-dependent negative modulators of membrane potential (21, 35, 38). They cause transient but large increases of [Ca2+]i in the subsarcolemmal spaces to activate nearby Ca2+-activated K+ channels and generate spontaneous transient outward currents (STOCs), leading to membrane hyperpolarization, reduction in Ca2+ influx through L-type Ca2+ channels, and vasodilation. It has been suggested that vasoconstrictors, such as norepinephrine, may enhance vasoconstriction by reducing Ca2+ spark frequency (3, 20, 30) to relieve the hyperpolarization imposed by Ca2+-activated K+ (KCa) channels; and vasodilators, such as nitric oxide and carbon monoxide, may potentiate vasorelaxation by enhancing spark frequency to activate STOCs (4, 19, 41, 49). Because of their highly localized and transient nature, Ca2+ sparks are thought to have little contribution to myofilament activation and smooth muscle contraction.

However, the regulation of vascular reactivity varies among different vascular beds because of their specialized physiological functions. The divergence reaches the extreme between systemic and pulmonary arteries. For example, pulmonary arteries have a much lower basal tone compared with systemic arteries and constrict instead of dilate in response to hypoxia. The functions and regulations of Ca2+ sparks in pulmonary and systemic arteries also appear to be different. Developmental studies showed that STOCs, hence Ca2+ sparks, are very active in fetal pulmonary artery smooth muscle cells (PASMCs), and their activity diminishes with maturation (39, 43). This is in marked contrast to the hundredfold increase in Ca2+ spark and STOC frequencies during maturation in systemic arterial myocytes (14). We have recently characterized Ca2+ sparks in rat intralobar PASMCs and found that, despite the fact that their spatiotemporal properties are similar to those of systemic myocytes, pulmonary Ca2+ sparks possess unique physiological properties. They are usually associated with membrane depolarization instead of hyperpolarization and are activated specifically by the vasoconstrictor endothelin-1 (ET-1) but not by norepinephrine, even though both elicited similar global Ca2+ transients (42). These findings raise the intriguing possibility that Ca2+ sparks may increase vasomotor tone and participate specifically in the ET-1-mediated contraction of pulmonary arteries. This notion bears special significance because ET-1 has been implicated as a major modulator or mediator of hypoxia-induced pulmonary hypertension. Antagonists of ET receptors have been shown to prevent and reverse acute and chronic hypoxia-induced pulmonary hypertension in intact animals (6, 10). Furthermore, a threshold concentration (0.1 nM) of ET-1, which itself did not cause an increase in global Ca2+ concentration ([Ca2+]) or contraction, was found to potentiate hypoxia-induced contraction in PASMCs and restore acute hypoxic constriction in endothelium-denuded distal pulmonary arteries (27, 45). Because of these important physiological implications, we sought to determine in the present study 1) the concentration-dependent effects of ET-1 on the frequency of occurrence and the spatiotemporal properties of Ca2+ sparks, 2) the contribution of Ca2+ sparks in ET-1-induced pulmonary vasoconstriction, and 3) the signal transduction mechanism through which ET-1 regulates Ca2+ sparks.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSIONS
 DISCLOSURES
 REFERENCES
 
Isolation and culture of PASMCs. PASMCs were enzymatically isolated and transiently cultured as previously described (47). The procedures involving animals have been reviewed and approved by the Johns Hopkins University Animal Care and Use Committee. Briefly, male Wistar rats (150-200 g) were injected with heparin and anesthetized with pentobarbital sodium (130 mg/kg ip). They were exsanguinated, and the lungs were removed and transferred to a petri dish filled with HEPES-buffered salt solution (HBSS) containing (in mM) 130 NaCl, 5 KCl, 1.2 MgCl2, 1.5 CaCl2, 10 HEPES, and 10 glucose, pH 7.4 (adjusted with NaOH). Second- and third-generation intrapulmonary arteries (~300-800 µm) were isolated and cleaned free of connective tissue. The endothelium was removed by gently rubbing the luminal surface with a cotton swab. Arteries were then allowed to recover for 30 min in cold (4°C) HBSS, followed by 20 min in reduced-Ca2+ (20 µM) HBSS at room temperature. The tissue was digested at 37° C for 20 min in 20 µM Ca2+ HBSS containing collagenase (type I, 1,750 U/ml), papain (9.5 U/ml), BSA (2 mg/ml), and dithiothreitol (1 mM), then removed, and washed with Ca2+-free HBSS to stop digestion. Single smooth muscle cells were dispersed gently by trituration with a small-bore pipette in Ca2+-free HBSS at room temperature. The cell suspension was then placed on 25-mm glass coverslips and transiently (16-24 h) cultured in Ham's F-12 medium (with L-glutamine) supplemented with 0.5% FCS, 100 U/ml streptomycin, and 0.1 mg/ml penicillin.

Measurement of Ca2+ sparks. Ca2+ sparks were visualized as previously described (42) using the membrane-permeable Ca2+-sensitive fluorescent dye fluo 3-AM. PASMCs were loaded with 5-10 µM fluo 3-AM (dissolved in DMSO with 20% pluronic acid) for 30-45 min at room temperature (~22° C) in normal Tyrode solution containing (in mM) 137 NaCl, 5.4 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, and 10 glucose, pH 7.4 (adjusted with NaOH). Cells were then washed thoroughly with Tyrode solution to remove extracellular fluo 3-AM and rested for 15-30 min in a cell chamber to allow for complete deesterification of cytosolic dye. Confocal images were acquired using a Zeiss LSM-510 inverted confocal microscope (Carl Zeiss) with a Zeiss Plan-Neofluor x40 oil immersion objective [numerical aperture (NA) = 1.3]. The confocal pinhole was set to render a spatial resolution of 0.4 µm in the x-y axis and 1.7 µm in the z-axis. Fluo 3-AM was excited by the 488-nm light of an argon laser, and fluorescence was measured at >505 nm. Images were acquired in the line scan mode (digital zoom rendering a 38-µm scan line), scanning at 0.075 µm/pixel, 512 pixel/line at 2-ms intervals for 512 lines/image from different cells within the same culture dish before and after drug application. Photobleaching and laser damage to the cells were minimized by attenuating the laser to ~1% of its maximum power (25 mW) with an acousto-optical tunable filter. Only 10 images (once every 10 s) were taken from each cell. Cells that did not respond to an external solution containing 10 mM Ca2+ and 0.5 mM caffeine applied at the end of experiments were discarded. All experiments were performed at room temperature.

Photolysis of caged inositol trisphosphate. PASMCs were loaded with 2 µM of a caged membrane-permeant derivative of inositol trisphosphate (IP3), ci-IP3/PM (L1-27), that is structurally similar to the original caged derivative of IP3 (cm-IP3) (26), except that the 2- and 3-hydroxyl groups of the inositol ring are protected as an acetonide (isopropylidene). Like cm-IP3/PM, ci-IP3/PM causes Ca2+ release from IP3R-gated stores upon ultraviolet (UV)-induced photolysis (Li, unpublished observation). It was dissolved in DMSO with 20% pluronic acid, mixed together with the Ca2+ indicator dye fluo 3-AM, and applied to PASMCs on coverslips for 30-45 min. After being washed, cells were incubated for another 20-30 min for deesterification before imaging. The caged IP3 was released by photolysis of the compound using an N2 pulse laser [model GL-3300 (PTI), maximum pulse energy of 2 mJ] at the wavelength of 337 nm. The laser pulse energy was attenuated to ~300 µJ and delivered to PASMCs using UV-enhanced fiber-optics (400 µm in diameter) positioned <50 µm from the myocytes. Only one photolysis was induced in each cell.

Isometric contraction. Intralobar pulmonary arteries (300-800 µm OD) were isolated and placed in oxygenated modified Krebs solution containing (in mM) 118 NaCl, 4.7 KCl, 0.57 MgSO4, 1.18 KH2PO4, 25 NaHCO3, 10 dextrose, and 2.5 CaCl2. They were cleaned of connective tissue and cut into 4-mm lengths. Endothelium was disrupted by gently rubbing the lumen with a small wooden stick, and the arterial rings were suspended between two stainless steel stirrups in organ chambers filled with modified Krebs solution for isometric tension recording. The solution was gassed with 16% O2-5% CO2 to maintain a pH of 7.4 and the temperature at 37°C. Isometric contraction was measured using a strain gauge connected to a Grass polygraph. The resting tension was adjusted to 1 g. The arteries were exposed to 80 mM KCl to established maximum contraction and to phenylephrine (3 x 10-7 M) followed by ACh (10-6 M) to verify complete disruption of endothelium.

Immunolocalization of RyRs and IP3Rs. PASMCs were fixed in 2% paraformaldehyde for 10 min. The cells were washed with 0.75% glycine in PBS for 30 min, followed by permeabilization with 0.05% Triton X-100 for 15 min. The cells were then incubated with a combination of unconjugated donkey anti-rabbit IgG, anti-mouse IgG, or anti-mouse IgM antibodies (1:20) to block nonspecific binding for 2 h and then incubated overnight with a combination of isoformspecific anti-RyR and anti-IP3R primary antibodies. Primary antibodies included rabbit polyclonal anti-IP3R type I, type II, and type III (1:100; Affinity Bioreagents); mouse monoclonal IgG anti-IP3R (1:100; Chemicon) and mouse monoclonal IgG anti-IP3R type III (1:50, Transduction Laboratory); mouse monoclonal IgG anti-RyR type II (1:100; Affinity Bioreagents); and mouse monoclonal IgM anti-RyR type I (1:100; Upstate). Antibody-specific binding was visualized with either donkey CY2-conjugated or CY5-conjugated secondary antibodies (1:100; Jackson Immunoresearch Laboratory), which were affinity purified for multiple labeling. BSA (0.2%) and Triton X-100 (0.05%) were included in PBS for antibody dilution. Incubation was carried out in a moistened chamber at 4°C. Immunolocalization was examined with an MRC-1000 confocal scanning unit (Bio-Rad) equipped with a krypton-argon laser, and images were acquired with a Zeiss plan-apochromat objective (x63, NA = 1.4). PASMCs without exposure to primary antibodies were used as a negative control.

Data analysis. Ca2+ spark detection was guided by an algorithm custom written using the Interactive Data Language software (Research Systems, Boulder, CO). The program identified Ca2+ sparks on the basis of their statistical deviation from background noise, similar to that described previously by Cheng et al. (8). Fluorescence signals (F) of each confocal image were first normalized in terms of F/F0, where F0 is the baseline fluorescence in a region of the image without Ca2+ sparks. The mean and variance ({sigma}2) of the normalized image were determined. Ca2+ sparks were then identified based on local fluorescence intensity greater than mean + 3{surd}{sigma}2. Amplitudes of the Ca2+ sparks were then calibrated to absolute [Ca2+] by a pseudoratio method (7) using the equation [Ca2+]i = (KD · R)/{[(KD/[Ca2+]rest) + 1] - R}, where R was F/F0, and dissociation constant (KD) of fluo 3 was 1.1 µM. Resting [Ca2+] ([Ca2+]rest), in the absence of ET-1, was assumed to be 100 nM, and the values in the presence of different concentrations of ET-1 were determined from separate groups of cells independently. The duration and spread (or width) of Ca2+ sparks were quantified as the full-duration half-maximum (FDHM) and full-width half-maximum (FWHM), respectively. The spark frequency of each cell was defined as the number of sparks detected per second in a scan line of 38 µm. In some images, large increases in global [Ca2+] or clustering of sparks rendered individual sparks indiscernible. These global [Ca2+] increases were not analyzed with the program. Throughout the paper, data are expressed as means ± SE. The number of cells is specified in the text. Statistical significance (P < 0.05) of the changes in spark characteristics was assessed by paired or unpaired Student's t-tests, one-way ANOVA with Newman-Keul's post hoc analyses, or nonparametric Mann-Whitney U-tests, wherever applicable.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSIONS
 DISCLOSURES
 REFERENCES
 
Characterization of ET-1-induced Ca2+ spark. Under steady-state conditions, ET-1 at a concentration between 0.1 and 10 nM elicited concentration-dependent increases in spark frequency from the control of 0.62 ± 0.08 s-1 (n = 61 cells) to a maximum of 2.24 ± 0.20 s-1 (n = 46 cells, P < 0.001) at 10 nM ET-1 in rat intralobar PASMCs (Fig. 1, A and B). The effect was apparent even at 0.1 nM as a twofold increase in spark frequency (1.34 ± 0.15 s-1, P < 0.001, n = 62 cells). Spark amplitude, expressed as {Delta}[Ca2+]i, was significantly increased at all concentrations of ET-1 tested (control: 43.7 ± 1.5; 10 nM ET-1: 71.4 ± 1nM, P < 0.001). Spark duration (FDHM) was also significantly prolonged at ET concentrations >=1 nM (control: 35.6 ± 1.6 ms; 10 nM ET-1: 66.4 ± 1.7 ms, P < 0.001). By contrast, the spatial spread (FWHM) was increased only slightly at the highest concentration (10 nM) of ET-1.



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Fig. 1. Concentration-dependent effects of endothelin (ET)-1 on the biophysical properties of Ca2+ spark in pulmonary artery smooth muscle cells (PASMCs). A: representative line scan images showing Ca2+ sparks in the absence and presence of different concentrations of ET-1. B-E: bar graphs summarizing the effects of ET-1 on spark frequency, duration [full-duration half-maximum (FDHM)], amplitude (expressed as {Delta}[Ca2+]), and spatial spread [full-width half-maximum (FWHM)] of Ca2+ sparks obtained from control PASMCs (61 cells and 482 sparks) and PASMCs exposed to 0.1 nM (62 cells and 831 sparks), 1 nM (64 cells and 1,037 sparks), 3 nM (61 cells and 1,272 sparks), and 10 nM (46 cells and 1,058 sparks) ET-1. *Significant difference from control, P < 0.05.

 

Since the distributions of spark amplitude, duration, and size are non-Gaussian because of the inherited property of off-center sampling in line scan confocal imaging (8, 40), nonparametric statistics were applied to further compare spark properties in the absence and presence of 3 nM ET-1 (Fig. 2). When compared with the Mann-Whitney U-test, the amplitude of Ca2+ sparks elicited by ET-1 was found to be significantly higher than that of control (control: median = 35.6 nM, 90% range = 20.3-94.4 nM; ET-1: median = 64.0 nM, 90% range = 35.8-140.7 nM; P < 0.001). Spark duration (FDHM) was significantly longer (control: median = 26 ms, 90% range = 14-80 ms; ET-1: median = 36 ms, 90% range = 16-108 ms; P < 0.001), whereas the spread (FWHM) was unaltered (control: median = 1.57 µm, 90% range = 0.9-2.88 µm; ET-1: median = 1.50 µm, 90% range = 0.6-3.2 µm) in the presence of ET-1. Therefore, both parametric and nonparametric analyses indicate that ET-1 caused dramatic increases in Ca2+ spark frequency, duration, and amplitude in rat PASMCs.



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Fig. 2. Frequency distributions of the spatiotemporal parameters of Ca2+ spark in the absence (A-C, left, 61 cells and 482 sparks) and presence (A-C, right, 61 cells and 1,272 sparks) of 3 nM ET-1. Nos. of events are expressed as the functions of spark amplitude (A), duration (FDHM, B), and spatial spread (FWHM, C). Medians of the frequency distributions were compared, and P values were determined by the Mann-Whitney U-test. NS, not significant.

 

Ca2+ sparks activated by ET-1 originated primarily from Ca2+ release via RyRs. Pretreatment of PASMCs with 50 µM ryanodine for 30 min almost completely abolished spontaneous Ca2+ sparks generated at rest, and in the presence of ET-1 (Fig. 3 A). Residual Ca2+ sparks were most likely the result of incomplete block of RyRs, instead of originating from another source, because the amplitude, duration, and size of these sparks were similar to those recorded in the absence of ryanodine.



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Fig. 3. Effects of ryanodine (ryan; 50 µM) on ET-1-induced spark frequency in PASMCs and contraction in intralobar pulmonary arteries. A: bar graph showing control and ET-1 (3 nM)-induced spark frequency recorded in PASMCs with or without pretreatment with 50 µM ryanodine (ryanodine = 62 cells, ET-1+ryanodine = 40 cells). B: concentration-tension curves of ET-1 obtained in isolated deendothelialized intralobar pulmonary arterial rings in the absence or presence of ryanodine (50 µM). *Significant difference between PASMCs or pulmonary arteries with or without pretreatment with ryanodine.

 

Ca21+ sparks and ET-1-induced contraction. The contribution of Ca2+ sparks to ET-1-induced vasoconstriction was evaluated in endothelium-denuded intralobar pulmonary arterial rings. ET-1 elicited a sustained increase in tension, beginning at 0.1 nM, and reached the maximum at 10 nM, with an EC50 of 0.88 ± 0.13 nM (n = 9; Fig. 3 B). Maximum tension elicited by ET-1 was about two times that elicited by 80 mM KCl (KCl: 0.33 ± 0.03 g; ET-1: 0.59 ± 0.05 g). Pretreatment with 50 µM ryanodine for 30 min increased the threshold concentration for contraction and shifted the concentration-response curve of ET-1 to the right (EC50 = 2.43 ± 0.19 nM, n = 10, P < 0.001) but had no apparent effect on the baseline or maximal tension.

Probing the mechanisms: L-type Ca2+ channels, sarcoplasmic reticulum Ca2+, and cADP-ribose. To explore the mechanism through which ET-1 enhances Ca2+ spark frequency, the common pathways for spark activation were examined. Inhibition of L-type Ca2+ channels with 10 µM nifedipine had no significant effect on spontaneous Ca2+ sparks in PASMCs at rest, or in the presence of 3 nM ET-1. Similarly, pretreatment of PASMCs with a membrane-permeant cADP-ribose (cADPR) antagonist, 8-bromo-cADPR (100 µM), for 20 min had no significant effect on resting spark frequency and failed to reduce spark frequency in the presence of ET-1.

Because an increase in sarcoplasmic reticulum (SR) luminal Ca2+ is known to enhance spark frequency, SR Ca2+ content was estimated by the exhaustive release of SR Ca2+ induced by a high concentration of caffeine. Rapid application of 10 mM caffeine for 10 s to PASMCs activated a large Ca2+ transient with a magnitude proportional to the amount of SR Ca2+ (Fig. 4B). The measurement was highly repeatable, as a Ca2+ transient of similar magnitude was consistently elicited when a second caffeine challenge was applied after a 15-min period of recovery. However, when PASMCs were pretreated with ET-1 (3 nM) after the first challenge, caffeine-induced Ca2+ transients were significantly reduced from an averaged peak [Ca2+] of 1.6 ± 0.3 to 0.8 ± 0.2 µM (n = 8, P < 0.05; Fig. 4, C and D). These results, therefore, suggest that activation of Ca2+ sparks by ET-1 was not mediated via activation of L-type Ca2+ channel, cADPR production, or an increase in SR Ca2+ content.



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Fig. 4. Lack of involvement of L-type Ca2+ channels, cADP-ribose, and sarcoplasmic Ca2+ content in ET-1-induced Ca2+ sparks. A: bar graph showing the resting and ET-1-evoked spark frequency in control PASMCs (61 cells) and myocytes pretreated with nifedipine (Nif, 10 µM, 25 cells) and 8-bromo (Br)-cADPR (100 µM, 35 cells). B: representative trace of repeated applications of caffeine (10 mM, 10 s) on intracellular Ca2+ concentration ([Ca2+]i). Similar results were obtained in 5 other cells. C: representative trace showing a reduction in the caffeine-induced Ca2+ transient after a PASMC was exposed to ET-1 (3 nM) for 10 min. D: bar graph showing the averaged data of peak [Ca2+]i induced by 10 mM caffeine before and after 10 min exposure to ET-1 (3 nM, n = 7 cells). *Significant difference from the value obtained before ET-1 exposure.

 

Signal transduction pathway for ET-1 activation of Ca2+ sparks. Application of the ETA receptor antagonist BQ-123 (1 µM) had no effect on the resting spark frequency (0.80 ± 0.11 s-1, n = 50 cells) but completely abolished Ca2+ sparks activated by ET-1 (3 nM; 0.76 ± 0.11 s-1, n = 52 cells; Fig. 5A). In contrast, the ETB receptor antagonist BQ-788 (1 µM) failed to inhibit the ET-1-induced Ca2+ sparks. U-73122 (100 nM), a specific PLC inhibitor, had no effect on the resting spark frequency but almost completely inhibited the ET-1-induced increase in spark frequency (1.11 ± 0.17 s-1, n = 50 cells; Fig. 5B). Moreover, caffeine at 0.5 mM caused a significant increase in spark frequency (1.66 ± 0.28 s-1, n = 16 cells) in the presence of U-73122, suggesting that the antagonist did not disrupt the ability of RyRs to generate Ca2+ sparks. Hence the increase in spark frequency was mediated exclusively through ETA receptor-dependent activation of PLC.



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Fig. 5. Pharmacological characterization of the signal transduction pathway mediating ET-1 stimulation of Ca2+ sparks in PASMCs. A: bar graph summarizing the effects of the ETA and ETB receptor antagonists, BQ-123 and BQ-788, respectively, on Ca2+ spark frequency in the absence and presence of 3 nM ET-1. B: effect of the PLC inhibitor U-73122 on Ca2+ spark frequency in the absence and presence of ET-1. C: effects of PKC inhibition by staurosporine (stau) on ET-1-enhanced spark frequency and PKC stimulation by 1-oleoyl-2-acetyl-sn-glycerol (OAG) on spark frequency. D: effects of inositol trisphosphate receptor (IP3R) antagonists 2-aminoethyl diphenylborate (2-APB) and xestospongin C (Xes C) on spark frequency in the presence or absence of ET-1. *Significant difference from ET-1-treated PASMCs in the absence of antagonists.

 

Activation of PLC increases the production of IP3 and diacylglycerol (DAG), the latter of which is the endogenous activator of PKC. To examine the PLC signaling cascade in the modulation of Ca2+ sparks, PASMCs were exposed to staurosporine (2 nM), a PKC inhibitor. Staurosporine had no effect on resting spark frequency and failed to prevent the ET-1 effect on Ca2+ sparks (Fig. 5C). Moreover, 1-oleoyl-2-acetyl-sn-glycerol (OAG, 100 µM), an analog of DAG, also failed to mimic the effect of ET-1. This excluded the involvement of DAG and PKC in the activation of Ca2+ spark by ET-1. By contrast, pretreatment of PASMCs with the IP3R antagonist 2-aminoethyl diphenylborate (2-APB; 50 µM, 20 min) caused a significant inhibition in the ET-1-induced increase in spark frequency (1.20 ± 0.18 s-1, n = 30 cells, P < 0.001 vs. control; Fig. 5D) without affecting the resting or caffeine (0.5 mM)-induced activation of spark frequency (1.72 ± 0.35 s-1, n = 23 cells). Moreover, another IP3R antagonist, xestospongin C (100 µM), also significantly inhibited the ET-1-induced Ca2+ sparks (1.08 ± 0.13 s-1, n = 35 cells). These results suggest that the activation of Ca2+ sparks by ET-1 is dependent on the availability of IP3Rs.

Photorelease of IP3 and Ca2+ sparks. To examine if IP3 generation could indeed directly activate Ca2+ sparks in PASMCs, the intracellular IP3 concentration was elevated by photolysis of a membrane-permeant caged IP3 analog, ci-IP3/PM. In a quiescent cell, uncaging IP3 caused an immediate activation of solitary Ca2+ sparks (Fig. 6A). In 26 cells, photorelease of IP3 caused an average increase in spark frequency from a control of 0.67 ± 0.33 s-1 to a peak of 4.44 ± 0.84 s-1 (P < 0.001) within 0.5 s after photolysis. In most cases, it was followed by a large global increase in [Ca2+] ({Delta}F/F0 = 1.14 ± 0.25, {Delta}[Ca2+]i = 167.5 ± 45.4 nM), at which Ca2+ sparks were no longer discernible (Fig. 6B). The spatiotemporal relationship between Ca2+ spark activation and global [Ca2+] increase is illustrated by a surface-plot of Ca2+ fluorescence recorded during a 1.5-s period immediately after photorelease of IP3 (Fig. 6C). The image clearly shows that small solitary Ca2+ sparks were activated before any noticeable increase in global [Ca2+]. These Ca2+ sparks then rapidly fused into larger clusters of local Ca2+ release and subsequently transformed into an avalanche of global Ca2+ release. Similar to the ET-1-induced Ca2+ sparks, IP3-induced Ca2+ sparks were completely abolished by 50 µM ryanodine (Fig. 4D). The global Ca2+ transient elicited by IP3 was also greatly reduced in the presence of ryanodine ({Delta}F/F0 = 0.51 ± 0.18, {Delta}[Ca2+]i = 70.8 ± 27.1 nM, n = 28, P < 0.05), suggesting that Ca2+ sparks constitute a major component of the IP3-induced global [Ca2+] transient. The residual global Ca2+ transient was completely obliterated by the IP3R antagonist 2-APB, indicating that the global and local Ca2+ release events were initiated via IP3Rs (Fig. 6E). Moreover, UV flashes alone did not trigger either local or global Ca2+ release in PASMCs without loading with the caged IP3 compound (n = 15, data not shown), suggesting that UV laser with pulse energy used had no immediate effect on intracellular Ca2+ mobilization.



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Fig. 6. Photolysis of the membrane-permeant caged IP3 analog ci-IP3/PM induced local and global changes of [Ca2+]i in PASMCs. A: line scan image showing a gradual activation of Ca2+ spark and global increase of [Ca2+]i after an ultraviolet (UV) flash in a PASMC loaded with ci-IP3/PM. B: line scan image representative of the average response to photolysis of ci-IP3/PM in control PASMCs. Bar graph shows the frequency distribution of Ca2+ sparks, and the red line-scattered plot shows the spatially averaged Ca2+ transient before and after the UV flash (n = 26 cells). C: three-dimensional pseudocolor surface plot generated from the image within the red box in B, showing the local Ca2+ events at the onset of a Ca2+ transient immediately after the photolysis of ci-IP3/PM. *Discernible Ca2+ spark before the global increase in Ca2+ concentration. D: composite containing a representative image; the group-averaged spark frequency distribution and the spatially averaged Ca2+ transients before and after photolysis of ci-IP3/PM in ryanodine-treated PASMCs (n = 27 cells). E: composite containing a representative image; the group-averaged spark frequency distribution and the spatially averaged Ca2+ transients before and after photolysis of ci-IP3/PM in 2-APB-treated PASMCs (n = 35 cells). F, fluorescence; F0, baseline fluorescence.

 

Immunolocalization of IP3Rs and RyRs. The localizations of IP3Rs and RyRs in PASMCs were examined further by immunohistocytochemistry. With the use of specific antibodies, two subtypes of RyR (RyR1 and RyR2) and three different subtypes of IP3R (IP3R1, IP3R2, and IP3R3) were detected in PASMCs. RyR1 and RyR2 were found in both sarcolemma and cytoplasm, and their distributions were very similar, as shown by the high degree of overlapping of their immunofluorescent signals (Fig. 7). Similar spatial distributions were also observed when RyR1 or RyR2 was doubled stained with another antibody raised against a common domain of IP3R1, IP3R2, and IP3R3. When antibodies specific to individual IP3R subtypes were used, IP3R3 exhibited a similar, whereas IP3R1 or IP3R2 showed a less consistent, pattern of localization as RyRs. These results provide morphological evidence in support of the notion that some IP3Rs and RyRs are closely associated in PASMCs.



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Fig. 7. Confocal images of immunostaining of ryanodine receptors (RyRs) and IP3Rs in PASMCs. A: double staining of RyR1 (red) and RyR2 (green) in PASMCs using isoform-specific monoclonal antibodies. Overlay, overlay images of RyR1 and RyR2 from the same cell; yellow indicates positive detection of both RyR isoforms in the same pixel. B: double staining of RyR1 and IP3R using a polyclonal antibody against a common domain of IP3R1, IP3R1, and IP3R3. C: double staining of RyR1 and IP3R3 using a monoclonal antibody specific for IP3R3.

 


    DISCUSSIONS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSIONS
 DISCLOSURES
 REFERENCES
 
ET-1, the most potent endothelial-derived vasoconstrictor, is known to exert its action through activation of ETA and ETB receptors, both of which are present abundantly in pulmonary arterial smooth muscle (25, 29). Binding of ET to its receptors triggers a wide spectrum of responses, including membrane depolarization, inhibition of voltage-gated K+ channels, activation of L-type Ca2+ current and nonselective cation channels, mobilization of intracellular Ca2+, and an increase in myofilament sensitivity (11, 13, 16, 18, 33,36, 46, 47). Depending on the type of vascular tissue, all or several of these mechanisms operate synergistically to cause vasoconstriction. In the present study, we have provided direct evidence suggesting that Ca2+ spark activation serves as a novel mechanism of ET-1-dependent vasoconstriction in pulmonary arteries.

ET-1 at 0.1-10 nM caused a concentration-dependent increase in the frequency, duration, and amplitude of Ca2+ sparks in PASMCs. The most dramatic effect was the increase in the apparent spark frequency, which was significant at the lowest concentration and reached fourfold at 10 nM ET-1. In fact, the actual spark frequency at higher ET-1 concentrations could be even higher because of the periodic occurrence of global increases in [Ca2+] and Ca2+ waves, which rendered Ca2+ sparks indiscernible. Ca2+ sparks activated by ET-1 originated exclusively from RyRs, as they were abolished by ryanodine, and contributed significantly to ET-1-induced pulmonary vasoconstriction. The rightward shift of the ET-1 concentration-tension relationship without a reduction in the maximum developed tension in the presence of ryanodine (Fig. 3B) suggests that Ca2+ sparks contribute to pulmonary vasoconstriction elicited by ET-1, with the highest efficiency at the low concentrations. Our previous study showed that Ca2+ sparks caused membrane depolarization in PASMCs (42). Such membrane depolarization induced by Ca2+ sparks may potentiate the effect of ET-1 on L-type Ca2+ channel activation (16, 17) by setting membrane potential close to their activation threshold and/or sustaining the elevation of cytoplasmic [Ca2+] by reducing Ca2+ removal through the Na+/Ca2+ exchanger. This modulatory mechanism is likely more effective at the lower than higher ET-1 concentrations because all vasoconstrictory mechanisms activated by ET-1 are operated optimally at the higher concentration. It is intriguing that ET-1 activated substantial Ca2+ sparks at 0.1 nM, a threshold concentration that hardly activated any change in global [Ca2+] and contraction. This is coincident with the fact that ET-1 at such concentration enhances contraction induced by other agonists through a process called "threshold potentiation" (34, 50) and potentiates dramatically the otherwise insignificant acute hypoxic pulmonary vasoconstriction in PASMCs and endothelium-denuded pulmonary microvessels (27, 45) through a process known as "priming." In these cases, Ca2+ sparks activated by ET-1 at the threshold concentration might have escaped detection by global [Ca2+]i measurement and exerted their priming effect through modulation of membrane potential. This interesting possibility needs further future investigations.

In addition to local Ca2+ signaling, Ca2+ sparks of PASMCs may also participate in vasoconstriction by providing Ca2+ for Ca2+/calmodulin activation of myofilaments. It is apparent in the caged IP3 experiments that PASMCs are capable of generating a large amount of Ca2+ sparks sufficient to elevate global [Ca2+]. It is also consistent with previous whole cell Ca2+ measurements showing that a major component of Ca2+ release induced by ET-1 could be blocked by ryanodine (47). In this case, ET-1-induced Ca2+ sparks of PASMCs are similar to the evoked Ca2+ sparks of striated muscles (5, 7, 28), underlying at least in part the global Ca2+ transients for PASMC contraction.

The notion that Ca2+ sparks potentiate the contraction of pulmonary arteries is in contrast to the well-documented vasodilatory effect in systemic arteries. In systemic vessels, inhibition of Ca2+ sparks with ryanodine causes vasoconstriction (21, 35, 38). Vasoconstrictors, such as norepinephrine and UTP, were found to cause a reduction in Ca2+ sparks (3, 20, 30). It has been proposed that vasoconstrictors shift Ca2+ signaling modalities from Ca2+ sparks to Ca2+ waves through the concerted actions of PKC to inhibit RyRs to reduce Ca2+ sparks and of IP3 to stimulate IP3Rs to generate Ca2+ waves. However, exceptions have been reported in portal vein myocytes in which norepinephrine and ANG II at low concentration activated Ca2+ sparks but at high concentration elicited Ca2+ waves (1, 2). Hence, PASMCs are more similar to portal vein myocytes in the regulation of Ca2+ sparks by vasoconstrictors, despite the differences that Ca2+ sparks are usually associated with STOCs in portal vein myoctyes (32) and norepinephrine does not induce Ca2+ sparks in PASMCs (42).

Our results clearly suggest that activation of Ca2+ sparks by ET-1 is mediated via ETA receptor-dependent activation of PLC. Activation of PLC generates IP3 and DAG, the latter of which activates multiple effectors through PKC-dependent phosphorylation, including RyRs and L-type Ca2+ channels (24, 31, 48). However, PKC-dependent phosphorylation is not a major pathway for Ca2+ spark activation, because staurosporine did not antagonize, and OAG did not mimic, the ET-1-induced activation of Ca2+ sparks. Moreover, inhibition of L-type Ca2+ channels with nifedipine was ineffective in ablating Ca2+ sparks elicited by ET-1. This is somewhat surprising because ET-1 has been shown to increase L-type Ca2+ current in systemic myocytes (16, 17), and activation of L-type Ca2+ channels is known to increase spark frequency in systemic and pulmonary myocytes (22, 35, 42). In addition, Ca2+ spark activation by ANG II in portal vein myocytes is mediated via L-type Ca2+ channels (1). The paradoxical lack of involvement of L-type Ca2+ channels in spark activation in PASMCs could be explained by the notions that L-type Ca2+ channels are loosely coupled or uncoupled to RyRs, a situation described in nonvascular myocytes (9), and that the activation of RyRs by ET-1 is dominated by another more efficacious mechanism. This mechanism, however, is neither related to an increase in SR Ca2+ content, because the caffeine-induced Ca2+ transient was in fact reduced in the presence of ET-1, nor related to cADPR production because 8-bromo-cADPR did not block the ET-1-induced change in spark frequency.

On the other hand, our results point unequivocally to the involvement of IP3-induced Ca2+ release. Two structurally distinctive antagonists of IP3R, 2-APB and xestospongin C, both inhibited almost completely the ET-1-induced Ca2+ sparks. The inhibition was not the result of nonspecific effects of the antagonists because the basal spark frequency and caffeine-induced Ca2+ sparks were unaltered. More convincingly, photorelease of IP3 directly triggered numerous Ca2+ sparks and large global Ca2+ transients, both of which were completely blocked by 2-APB. These results suggest that IP3 generation alone is sufficient to account for the activation of Ca2+ sparks, independent of other ET-1-related mechanisms. The absence of "Ca2+ puffs" induced by IP3 in the presence of ryanodine in rat PASMCs is consistent with previous reports in systemic and canine pulmonary myocytes that Ca2+ release from IP3Rs mainly generates Ca2+ waves and/or global Ca2+ transients (2, 20, 23), presumably because of the lack of clustering of IP3Rs in vascular myocytes.

Ca2+ release from IP3Rs may activate Ca2+ sparks by Ca2+-induced Ca2+ release (12) through elevation of global [Ca2+] or local [Ca2+] in the vicinity of RyRs. Confocal images revealed that IP3 activated solitary Ca2+ sparks, which intensified and merged to generate global Ca2+ transients, with no noticeable increase in global [Ca2+] before spark activation (Fig. 6C). This observation suggests that the initial activation of Ca2+ sparks might be the result of local Ca2+ signaling between closely associated IP3Rs and RyRs, a scenario similar to the functional coupling between KCa channels and RyRs in cerebral arteries (21) and between L-type Ca2+ channels and RyRs in cardiac myocytes (44). In this case, opening of one or a few IP3Rs generates microdomains of large [Ca2+] gradient to activate coordinated Ca2+ release from a spatially associated RyR cluster and generate Ca2+ sparks. Spatial association between RyRs and IP3Rs is supported at least circumstantially by immunostaining, which shows that multiple subtypes of RyRs (RyR1 and RyR2) and IP3Rs (IP3R1, IP3R2, and IP3R3) are present in PASMCs, and RyRs and IP3Rs exhibit similar distribution patterns in PASMCs (Fig. 4). Local interactions of the release channels have recently been proposed in portal vein myocytes (2, 15). In these myocytes, IP3Rs and RyRs were found to share a common Ca2+ pool (37), and spontaneous Ca2+ sparks of large and small sizes generated at a single frequent discharge site (15). It has been postulated that IP3 produced by the basal activity of PLC activates IP3Rs to recruit neighboring clusters of RyRs within the frequent discharge site to generate large Ca2+ sparks (15). During agonist stimulation, enhanced production of IP3 causes large-scale cross-activation of IP3Rs and RyRs to generate Ca2+ waves (2). In contrast, IP3Rs and RyRs have been shown to regulate separate Ca2+ pools in canine PASMCs (23). Ca2+ sparks of rat and canine PASMCs have a size similar to the small regular Ca2+ sparks observed in portal veins, and their occurrence is random with no clear indication of a frequent discharge site (23, 42). Additionally, inhibition of IP3Rs with 2-APB or xestospongin C had no effect on the resting frequency, size, and amplitude of spontaneous Ca2+ sparks (Fig. 5D), suggesting that IP3Rs do not contribute to the basal generation of Ca2+ sparks in rat PASMCs. Nevertheless, despite the differences from portal vein myocytes, our results clearly suggest that cross-activation of RyRs by Ca2+ released from IP3Rs did occur in PASMCs. It is apparent both when endogenous generation of IP3 was activated by ET-1 and when exogenous IP3 was introduced by flash photolysis. In view of the similar immunostaining patterns of RyRs and IP3Rs, it is possible that some RyRs may reside close enough to be activated by the Ca2+ microdomains of IP3Rs. Such functional coupling between IP3Rs and RyRs may provide a highly efficient amplification mechanism for pharmacomechanical coupling in pulmonary arteries to mobilize Ca2+ simultaneously from IP3R- and RyR-gated stores upon agonist stimulation.

In conclusion, our present study provides definitive evidence that ET-1 enhances Ca2+ spark generation from RyRs in PASMCs. The process is mediated through the ETA receptor-PLC-IP3 pathway but is unrelated to SR Ca2+ loading, Ca2+ influx via L-type Ca2+ channels, generation of cADPR, and activation of PKC. Moreover, IP3Rs and RyRs are coupled functionally and associated spatially in PASMCs. This local Ca2+ signaling process between IP3Rs and RyRs constitutes a novel mechanism that contributes significantly to ET-1-induced pulmonary vasoconstriction.


    DISCLOSURES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSIONS
 DISCLOSURES
 REFERENCES
 
This work was supported in part by National Heart, Lung, and Blood Institute Grants HL-071835 and HL-63813 and an American Heart Association (AHA; National Center) Grant-in-Aid to J. S. K. Sham. W. H. Li was supported by a research grant (I-1510) from the Robert A. Welch Foundation and a Career Development Award from the American Diabetes Association. W. M. Zhang was a visiting fellow from Liu Hua Qiao Hospital, Peoples Republic of China and was supported by a Postdoctoral Fellowship Award from the AHA (Mid-Atlantic Affiliate).


    FOOTNOTES
 

Address for reprint requests and other correspondence: J. S. K. Sham, Div. of Pulmonary and Critical Care Medicine, Johns Hopkins Asthma and Allergy Center, 5501 Hopkins Bayview Circle, Baltimore, MD 21224 (E-mail: jsks{at}welchlink.welch.jhu.edu).

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. Section 1734 solely to indicate this fact.


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 ABSTRACT
 MATERIALS AND METHODS
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 DISCUSSIONS
 DISCLOSURES
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