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Am J Physiol Lung Cell Mol Physiol 293: L674-L685, 2007. First published June 15, 2007; doi:10.1152/ajplung.00141.2007
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Ca2+ signaling in hypoxic pulmonary vasoconstriction: effects of myosin light chain and Rho kinase antagonists

Jian Wang, Letitia Weigand, Joshua Foxson, Larissa A. Shimoda, and J. T. Sylvester

Division of Pulmonary and Critical Care Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland

Submitted 9 April 2007 ; accepted in final form 12 June 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Antagonists of myosin light chain (MLC) kinase (MLCK) and Rho kinase (ROK) are thought to inhibit hypoxic pulmonary vasoconstriction (HPV) by decreasing the concentration of phosphorylated MLC at any intracellular Ca2+ concentration ([Ca2+]i) in pulmonary arterial smooth muscle cells (PASMC); however, these antagonists can also decrease [Ca2+]i. To determine whether MLCK and ROK antagonists alter Ca2+ signaling in HPV, we measured the effects of ML-9, ML-7, Y-27632, and HA-1077 on [Ca2+]i, Ca2+ entry, and Ca2+ release in rat distal PASMC exposed to hypoxia or depolarizing concentrations of KCl. We performed parallel experiments in isolated rat lungs to confirm the inhibitory effects of these agents on pulmonary vasoconstriction. Our results demonstrate that MLCK and ROK antagonists caused concentration-dependent inhibition of hypoxia-induced increases in [Ca2+]i in PASMC and HPV in isolated lungs and suggest that this inhibition was due to blockade of Ca2+ release from the sarcoplasmic reticulum and Ca2+ entry through store- and voltage-operated Ca2+ channels in PASMC. Thus MLCK and ROK antagonists might block HPV by inhibiting Ca2+ signaling, as well as the actin-myosin interaction, in PASMC. If effects on Ca2+ signaling were due to decreased phosphorylated myosin light chain concentration, their diversity suggests that MLCK and ROK antagonists may have acted by inhibiting myosin motors and/or altering the cytoskeleton in a manner that prevented achievement of required spatial relationships among the cellular components of the response.

isolated rat lung; pulmonary vascular resistance; angiotensin II; vascular smooth muscle; calcium channels

HYPOXIC PULMONARY VASOCONSTRICTION (HPV) is triggered by an increase in intracellular Ca2+ concentration ([Ca2+]i) in pulmonary arterial smooth muscle cells (PASMC) caused by Ca2+ release from the sarcoplasmic reticulum (SR) and Ca2+ entry through voltage-operated Ca2+ channels (VOCC) (57, 71). Recently, we and others demonstrated that Ca2+ influx through store-operated Ca2+ channels (SOCC) is required for HPV (42, 66, 77). SOCC are activated by depletion of SR Ca2+ stores (44, 45). The resulting Ca2+ influx, known as capacitative Ca2+ entry (CCE), is an important determinant of [Ca2+]i in a wide variety of vasomotor responses (19, 35, 50, 54).

Numerous studies in smooth muscle indicate that, at high concentrations, Ca2+ forms complexes with calmodulin, which activate myosin light chain (MLC) kinase (MLCK), causing phosphorylation of MLC (29). Phosphorylated MLC (P-MLC) then facilitates stimulation of myosin ATPase activity by actin, leading to cross-bridge cycling and contraction. Evidence indicates that this sequence of events occurs during HPV. Hypoxia increased MLC phosphorylation in PASMC and pulmonary arteries in association with contraction (33, 38, 68, 69, 83). In pulmonary arteries, tension development during hypoxia correlated directly with P-MLC concentration ([P-MLC]) and was abolished by the MLCK antagonist ML-9 (83).

[P-MLC] can be increased not only by activation of MLCK, but also by inhibition of myosin phosphatase (MP), the enzyme that dephosphorylates P-MLC (28, 29). In vascular smooth muscle, the principal regulator of MP appears to be Rho kinase (ROK). ROK is activated by the GTP-complexed form of the monomeric GTPase, Rho, which is produced when G protein-linked receptors interact with their ligands (52, 56). ROK inhibits MP by phosphorylating its myosin-binding subunit and/or by activating an endogenous inhibitor known as CPI-17 (17). ROK may also phosphorylate MLC directly (31). By these means, ROK can increase [P-MLC] at a given rate of MLC phosphorylation via MLCK. Because MLCK is activated by Ca2+-calmodulin complexes produced in proportion to [Ca2+]i, activation of ROK is said to increase myofilament Ca2+ sensitivity, defined as an increase in contractile force at a given [Ca2+]i (17, 53).

In rat distal pulmonary arteries, the slowly developing phase 2 of hypoxic contraction was associated with an elevated, but constant, [Ca2+]i (46). Hypoxia increased ROK activity in PASMC, and this increase was blocked by exoenzyme C3 or toxin B, antagonists of Rho (68). Hypoxia increased phosphorylation of MLC and the myosin-binding subunit of MP and decreased MP activity in PASMC (69). Y-27632 and HA-1077, antagonists of ROK, blocked hypoxia-induced MP inactivation and MLC phosphorylation in PASMC, phase 2 hypoxic contraction in distal pulmonary arteries, and HPV in isolated lungs and intact animals (16, 39, 47, 68, 69). Taken together, these results suggest that HPV requires an increase in Ca2+ sensitivity, as well as [Ca2+]i, and that the increase in Ca2+ sensitivity is mediated by Rho-activated ROK.

In several cell types, inhibitors of ROK and MLCK have altered increases in [Ca2+]i induced by a variety of agonists. For example, Y-27632 blocked [Ca2+]i responses to norepinephrine in rat mesenteric artery and rat aorta (18). Similarly, HA-1077 blocked increases in [Ca2+]i induced by norepinephrine in rat aorta (18, 59) and by bradykinin or thapsigargin in porcine aortic endothelial cells (72). ML-9 or a related MLCK antagonist, ML-7, blocked [Ca2+]i responses to KCl in coronary artery (80); methacholine, thapsigargin, or KCl in guinea pig trachealis (23); bradykinin, thapsigargin, or shear stress in endothelial cells (43, 58, 72, 73); and UTP in bovine adrenocortical fasciculata cells (30). In endothelial and adrenocortical fasciculata cells, isolated trachealis, and human blood monocytes, ML-9 blocked CCE through SOCC (23, 30, 43, 61). In portal vein myocytes, ML-7 and ML-9 blocked norepinephrine-induced increases in currents through nonspecific cation channels (2). These results suggest that, in addition to inhibition of the actin-myosin interaction, antagonists of MLCK and ROK can reduce [Ca2+]i through effects on Ca2+ channel activation.

It is not known whether MLCK and ROK antagonists alter Ca2+ signaling during HPV, which requires Ca2+ release from the SR and Ca2+ entry through VOCC and SOCC; therefore, we measured the effects of ML-9, ML-7, Y-27632, and HA-1077 on [Ca2+]i, Ca2+ entry, and Ca2+ release in rat distal PASMC exposed to hypoxia or depolarizing concentrations of KCl. Parallel experiments were performed in isolated rat lungs to determine the effects of these agents on pulmonary vasoconstriction.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
PASMC

As previously described (65, 66), distal (>4th generation) intrapulmonary arteries were dissected from lungs of pentobarbital sodium (65 mg/kg ip)-anesthetized male Wistar rats (300–500 g body wt). The luminal surfaces were rubbed with a cotton swab for removal of endothelial cells from the vascular segments. Smooth muscle cells were obtained by enzymatic digestion and cultured for 3–6 days on glass coverslips in Smooth Muscle Growth Medium 2 (Clonetics, Walkersville, MD) containing 5% serum in a humidified atmosphere of 5% CO2-95% air at 37°C. Serum concentration in the medium was decreased to 0.3% 24 h before an experiment to stop cell growth. Cellular purity was >95%, as assessed by morphological appearance under phase-contrast microscopy and immunofluorescence staining for {alpha}-actin (65, 66).

For measurement of [Ca2+]i and Ca2+ entry, cells were incubated with 7.5 µM fura 2-AM (Molecular Probes, Eugene, OR) for 60 min at 37°C under an atmosphere of 5% CO2-95% air. Coverslips were then mounted in a closed polycarbonate chamber clamped to a heated aluminum platform (model PH-2, Warner Instrument, Hamden, CT) on the stage of a inverted microscope (TSE 100 Ellipse, Nikon, Melville, NY) and perfused at 0.5 ml/min with Krebs-Ringer bicarbonate solution (KRBS), which contained (in mM) 118 NaCl, 4.7 KCl, 2.5 CaCl2, 0.57 MgSO4, 1.18 KH2PO4, 25 NaHCO3, and 10 glucose. Perfusate was equilibrated in heated reservoirs with 5% CO2 and either 16% O2 (normoxia) or 4% O2 (hypoxia) and led to the chamber through stainless steel tubing. For depolarization of the cells, perfusate KCl was increased to 60 mM and NaCl was decreased to 62.7 mM. Chamber temperature was maintained at 37°C with an in-line heat exchanger and dual-channel heater controller (models SF-28 and TC-344B, Warner Instrument).

After removal of extracellular dye by 10 min of normoxic perfusion, [Ca2+]i was determined at 6- to 60-s intervals from the ratio of fura 2 fluorescence emitted at 510 nm after excitation at 340 nm to that after excitation at 380 nm (F340/F380) measured in 10–30 cells using a xenon arc lamp, interference filters, an electronic shutter, a x20 fluorescence objective, and a cooled charge-coupled device imaging camera. Data were collected online with InCyte software (Intracellular Imaging, Cincinnati, OH). [Ca2+]i was estimated from F340/F380 measured in vitro in calibration solutions with 0–1,350 nM Ca2+ (Molecular Probes). After a 5-min control period, cells were exposed to 4% O2 for 20 min or 60 mM KCl for 15 min followed by a 10-min normoxic recovery period, during which perfusate KCl concentration ([KCl]) was normal. During exposure to hypoxia or high K+ concentration ([K+]), antagonists of MLCK (ML-9 and ML-7) or ROK (Y-27632 and HA-1077) were added to the perfusate in amounts sufficient to achieve the desired concentrations: 10, 30, or 100 µM ML-9 (n = 4/group), 1, 10, or 100 µM ML-7 (n = 4–5/group), 1, 3, or 10 µM Y-27632 (n = 4–5/group), and 10, 30, or 100 µM HA-1077 (n = 4–5/group). In each experiment, cells were exposed to hypoxia or high [K+] and treated with a single concentration of a single antagonist. Untreated cells from the same isolate served as controls (n = 4–9).

To assess CCE, we perfused PASMC with normoxic (16% O2) or hypoxic (4% O2) Ca2+-free KRBS containing 0.5 mM EGTA to chelate residual Ca2+, 5 µM nifedipine to prevent Ca2+ entry through L-type VOCC, and 10 µM cyclopiazonic acid (CPA) to deplete SR Ca2+ stores. In our first series of experiments, we estimated CCE after 10 min by measuring the increase in [Ca2+]i caused by restoration of perfusate [Ca2+] to 2.5 mM in cells exposed to 10, 30, or 100 µM ML-9 (n = 4–5/group), 1, 3, or 10 µM Y-27632 (n = 4–5/group), or 10, 30, or 100 µM HA-1077 (n = 3–4/group). Antagonists were given simultaneously with CPA. Untreated cells from the same isolate served as controls (n = 4–10). In our second series of experiments, we estimated CCE after 10 min by measuring the rate at which 200 µM MnCl2 quenched fura 2 fluorescence excited at 360 nm over the ensuing 10 min in untreated cells (n = 3 during normoxia and n = 4 during hypoxia) and cells exposed to hypoxia + ML-9 (100 µM, n = 4) or hypoxia + Y-27632 (10 µM, n = 4).

To assess Ca2+ release from SR, we perfused PASMC with normoxic Ca2+-free KRBS containing 0.5 mM EGTA to chelate residual Ca2+. After 10 min, the cells were perfused with hypoxic (4% O2) Ca2+-free KRBS for 5 min. We estimated Ca2+ release as the maximum increase in [Ca2+]i caused by hypoxia in PASMC exposed to 100 µM ML-9 (n = 4) or 10 µM Y-27632 (n = 4), which were added to the perfusate 8 min before hypoxia. Untreated cells served as controls (n = 4).

To assess voltage-operated Ca2+ entry, we perfused PASMC with normoxic Ca2+-free KRBS containing 0.5 mM EGTA to chelate residual Ca2+, 50 µM SKF-96365 to prevent CCE (65), and 60 mM KCl to cause depolarization. After 10 min, we estimated voltage-operated Ca2+ entry from the rate at which 200 µM MnCl2 quenched fura 2 fluorescence in PASMC exposed to 100 µM ML-9 (n = 3) or 5 µM nifedipine (n = 2). Untreated cells served as controls (n = 3).

Isolated Lungs

Male Wistar rats (200–400 g body wt) were given heparin (1,000 U ip) and anesthetized 20–30 min later with pentobarbital sodium (65 mg/kg ip). A tracheostomy was performed, and the animal was ventilated with room air at a tidal volume of 10 ml/kg and rate of 30 min–1 (rodent ventilator 883, Harvard Apparatus, Holliston, MA). After exsanguination from the femoral artery, the ventilating gas was changed to 16% O2-5% CO2. A thoracotomy was performed, and cannulas were inserted into the main pulmonary artery and left atrium, which drained into a heated reservoir. The lungs were perfused with a peristaltic pump (Ismatec Reglo Analog Pump, Cole Parmer, Vernon Hills, IL) at 40 ml·kg–1·min–1 with KRBS containing (in mM) 118 NaCl, 4.7 KCl, 0.57 MgSO4, 1.18 KH2PO4, 25 NaHCO3, and 10 glucose. Ficoll (4 g/dl) and sodium meclofenamate (3.1 µM) were added to provide oncotic pressure and prevent release of vasodilator prostaglandins. After the vasculature was flushed free of residual blood, the perfusate was recirculated. In KCl experiments, lungs were perfused with a 70:30 mixture of KRBS and the animal's own blood to mitigate edema formation caused by high perfusion pressures. Temperature in the left atrial effluent was maintained at 37°C with a heat exchanger. Pulmonary arterial pressure (Ppa), left atrial pressure, and tracheal pressure were measured relative to the bottom of the lung with pressure transducers (model P10EZ, Spectramed, Oxnard, CA) and recorded with a computer-linked recording system (Powerlab, ADInstruments, Colorado Springs, CO). End-expiratory tracheal and left atrial pressures were maintained at 3–4 and <0mmHg, respectively. Since perfusate flow was also constant, increases in Ppa were assumed to reflect pulmonary vasoconstriction.

After a 20-min stabilization period, lungs were subjected to one of two protocols: 1) four alternating exposures to ANG II (0.05-µg bolus into the main pulmonary artery) and hypoxia (ventilation with 2% O2 for 5 min) at 5- to 10-min intervals or 2) progressive 5–10 mM increases in perfusate [KCl] at 5- to 10-min intervals until Ppa achieved a maximum or the lungs developed edema. In protocol 1, antagonists of MLCK (30 or 100 µM ML-9 or ML-7) or ROK (3, 10, 30, or 100 µM Y-27632 or HA-1077) were added to the perfusate after the second hypoxic exposure (n = 4–6 at each antagonist concentration). In protocol 2, the antagonists (10, 30 or 100 µM ML-9, ML-7, Y-27632, or HA-1077; n = 4–5 at each antagonist concentration) were given 10–15 min before administration of KCl. In both protocols, a lung was exposed to a single concentration of a single antagonist. Untreated lungs served as controls (n = 9–10).

Drugs and Materials

Stock solutions of Y-27632, HA-1077, SKF-96935, and KCl were made up in water and stored at 4°C. Stock solutions of ML-9, ML-7, and ANG II were made up in water on the day of the experiment. Stock solutions of nifedipine and CPA were made up in DMSO and stored at –20°C. ML-9, ML-7, Y-27632, and nifedipine were obtained from CalBiochem (La Jolla, CA). All other agents were obtained from Sigma Chemical (St. Louis, MO).

Data Analysis

To estimate the concentrations at which MLCK or ROK antagonists inhibited responses by 50% (IC50), the mean {Delta}[Ca2+]i or maximum {Delta}Ppa elicited by hypoxia or KCl in antagonist-treated preparations was expressed as a percentage of the corresponding mean values in untreated preparations. To obtain IC50, we used an iterative least-squares method to fit the relationship between percent response and antagonist concentration to the Hill equation (66, 77). On two occasions in PASMC, mean {Delta}[Ca2+]i at the high antagonist concentration was either negative (Fig. 1, A and B : ML-7) or plateaued at >0 nM (Fig. 2, A and B : Y-27632, hypoxia). In these cases, average {Delta}[Ca2+]i at that concentration was subtracted from all observations before data were expressed as percentage of control.


Figure 1
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  		Fig. 1. Effects of myosin light chain (MLC) kinase (MLCK) antagonists (ML-9 and ML-7) and Rho-kinase antagonists (Y-27632 and HA-1077) on [Ca2+]i responses to hypoxia in rat distal pulmonary arterial smooth muscle cells (PASMC). A: time course of intracellular Ca2+ concentration ([Ca2+]i) at different antagonist concentrations. B: average change in [Ca2+]i during 3–23 min of hypoxic exposure in A in untreated control PASMC and cells treated with MLCK and ROK antagonists. C: concentration-response relations determined by least-squares iterative fit of Hill equation to mean data in B, expressed as percentages of values measured in control cells.

 

Figure 2
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  		Fig. 2. Effects of antagonists of MLCK (ML-9) and ROK (Y-27632 and HA-1077) on [Ca2+]i responses to restoration of extracellular Ca2+ concentration ([Ca2+]) to 2.5 mM during normoxia (N) and hypoxia (H) in rat distal PASMC perfused with Ca2+-free Krebs-Ringer bicarbonate solution (KRBS) containing 0.5 mM EGTA, 5 µM nifedipine (NIF), and 10 µM cyclopiazonic acid (CPA). A: time course of [Ca2+]i at different antagonist concentrations. B: maximum increase in [Ca2+]i after restoration of extracellular [Ca2+] in untreated control PASMC and cells treated with MLCK and ROK antagonists. Concentrations of ROK antagonists that caused maximum inhibition during hypoxia (H) had no effect during normoxia (N). C: concentration-response relations determined by least-squares iterative fit of Hill equation to mean data in B, expressed as percentages of values measured in control cells.

 
In PASMC isolates from a specific animal, we measured [Ca2+]i in each cell and then entered the average of these values into statistical analysis as an n of 1. Thus, in PASMC and isolated lung experiments, n refers to the number of animals. Statistical analyses were performed using Student's t-test or ANOVA. If a significant interaction F-ratio was obtained with the latter, pair-wise comparison of individual means was performed by calculating the least significant difference. Comparisons of group means with control were performed using Dunnett's test. Differences were considered significant when P < 0.05. Values are means ± SD unless otherwise stated.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Responses to Hypoxia

PASMC. Hypoxia caused rapid increases in PASMC [Ca2+]i that were sustained for the duration of exposure (Fig. 1A). ML-9 and ML-7, antagonists of MLCK, caused concentration-dependent inhibition of this response (Fig. 1, A and B), with estimated IC50 of 6.3 and 10.8 µM, respectively (Fig. 1C). Y-27632 and HA-1077, antagonists of ROK, also inhibited [Ca2+]i responses to hypoxia in a concentration-dependent manner (Fig. 1, A and B). Estimated IC50 was 2.5 µM for Y-27632 and 20.2 µM for HA-1077 (Fig. 1C).

Our previous studies indicated that the [Ca2+]i response to hypoxia in PASMC required CCE through SOCC (65, 66). To determine whether inhibition of this response by antagonists of MLCK and ROK was due to blockade of CCE, we determined the effects of ML-9, Y-27632, and HA-1077 on [Ca2+]i responses to restoration of extracellular Ca2+ in PASMC perfused with Ca2+-free KRBS containing CPA to deplete Ca2+ stores in the SR and nifedipine to prevent Ca2+ entry through VOCC (Fig. 2). Administration of CPA in the absence of extracellular Ca2+ caused a transient increase in [Ca2+]i, which was presumably due to Ca2+ release from the SR followed by Ca2+ uptake into intracellular storage sites not dependent on SR Ca2+-ATPase, such as mitochondria, and/or Ca2+ efflux via plasma membrane Ca2+-ATPase or Na+/Ca2+ exchange (Fig. 2A). The maximum increase in [Ca2+]i induced by CPA in the absence of extracellular Ca2+ averaged 126 ± 66 nM in normoxic control PASMC and was not altered by hypoxia or antagonists of MLCK and ROK. In contrast (Fig. 2), the maximum increase in [Ca2+]i caused by subsequent restoration of extracellular Ca2+ was markedly enhanced by hypoxia, as we observed previously (66). Moreover, this enhancement was abolished by ML-9, Y-27632, or HA-1077 (IC50 = 6.4, 1.2, and 23.9 µM, respectively; Fig. 2C). These IC50 values were virtually identical to those estimated for [Ca2+]i responses to hypoxia (6.3, 2.5, and 20.2 µM, respectively; Fig. 1C). To confirm that these effects were due to inhibition of Ca2+ influx through SOCC, rather than enhancement of Ca2+ efflux or uptake, we measured the effects of ML-9 and Y-27632 on quenching of fura 2 fluorescence by Mn2+ in PASMC perfused with Ca2+-free KRBS containing CPA and nifedipine (Fig. 3). Hypoxia increased the rate at which Mn2+ quenched fura 2 fluorescence more than twofold. This increase was eliminated by ML-9 (100 µM) or Y-27632 (10 µM).


Figure 3
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  		Fig. 3. Effects of antagonists of MLCK (100 µM ML-9) and ROK (10 µM Y-27632) on capacitative Ca2+ entry, measured as quenching of fura 2 fluorescence at 360 nm (F360) by 200 µM MnCl2 in PASMC perfused with Ca2+-free KRBS containing 0.5 mM EGTA, 5 µM nifedipine, and 10 µM CPA. A: time course of F360 expressed as a percentage of its value at time 0, the beginning of MnCl2 exposure. B: mean quenching of fura 2 fluorescence at 10 min calculated as 100 – F360 at 10 min, where F360 is expressed as a percentage of its value at time 0.

 
The MLCK antagonist ML-9 also inhibited CCE during normoxia (Fig. 2). The IC50 for this effect (5 µM) was equivalent to that observed for ML-9 vs. hypoxia-induced increases in CCE (6.4 µM; Fig. 2C) or [Ca2+]i (6.3 µM; Fig. 1C). In contrast, neither ROK antagonist (Y-27632 nor HA-1077) altered CCE during normoxia (Fig. 2).

In untreated control PASMC perfused with Ca2+-free KRBS, hypoxia caused a transient increase in [Ca2+]i (128 ± 19 nM), indicating intracellular Ca2+ release (Fig. 4). This increase was virtually abolished in cells treated with 100 µM ML-9 or 10 µM Y-27632 (17 ± 14 and 18 ± 11 nM, respectively, P < 0.0001). Baseline [Ca2+]i under normoxic Ca2+-free conditions averaged 106 ± 52 nM, did not differ among groups, and was not altered by ML-9 or Y-27632.


Figure 4
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  		Fig. 4. Effects of antagonists of MLCK (ML-9) and ROK (Y-27632) on intracellular release of Ca2+ induced by hypoxia in PASMC perfused with Ca2+-free KRBS containing 0.5 mM EGTA. A: time course of mean [Ca2+]i in untreated control PASMC and cells treated with ML-9 or Y-27632. Baseline [Ca2+]i under normoxic Ca2+-free conditions averaged 106 ± 15 nM and did not differ among groups. B: mean maximum increases in [Ca2+]i determined by measuring maximum value during hypoxia for each cell in an experiment, averaging these values across cells, and entering this average value as an n of 1 in the calculation of group means.

 
Isolated lungs. Figure 5 shows representative recordings of four alternating exposures to ANG II and hypoxia in untreated control lungs and lungs treated with an antagonist of MLCK (ML-9) or ROK (HA-1077) after the second exposure. Before treatment, ANG II and hypoxia caused rapid reversible increases in Ppa at constant flow, left atrial pressure, and end-expiratory tracheal pressure, indicating vasoconstriction. Peak {Delta}Ppa from baseline caused by hypoxia at the second exposure averaged 10.6 ± 1.3 mmHg in control lungs and did not differ among groups. Peak {Delta}Ppa at subsequent exposures, expressed as a percentage of its value at the second exposure, is shown in Fig. 6A. In control lungs, hypoxic pressor responses decreased slightly during the third and fourth exposures but remained >80% of the response at the second exposure. Antagonists of MLCK (ML-9 and ML-7) and ROK (Y-27632 and HA-1077) caused marked inhibition of pressor responses to hypoxia. Estimated IC50 values for ML-9, ML-7, Y-27632, and HA-1077 vs. hypoxic pressor responses were 29.0, 27.7, 31.2, and 54.9 µM, respectively (Fig. 6B). These values were 0.4–1 log unit greater than IC50 values vs. [Ca2+]i responses to hypoxia in PASMC (6.3, 10.8, 2.5, and 20.2 µM, respectively; Fig. 1C). Effects on pressor responses to ANG II were similar (data not shown).


Figure 5
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  		Fig. 5. Representative recordings of pulmonary arterial pressure (Ppa) in isolated rat lungs during 4 alternating exposures to ANG II (A, 0.05 µg bolus into the main pulmonary artery) and hypoxia (H, ventilation with 2% O2 for 5 min) in control lungs (A) and lungs treated with an MLCK antagonist (B; ML-9) or an ROK antagonist (C; HA-1077) after the 2nd exposure to ANG II and hypoxia.

 

Figure 6
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  		Fig. 6. Effects of antagonists of MLCK (ML-9 and ML-7) and ROK (Y-27632 and HA-1077) on maximum pulmonary arterial pressor responses to hypoxia ({Delta}Ppa) in isolated rat lungs. A: mean {Delta}Ppa during the 3rd and 4th exposures, measured as a percentage of values during the 2nd exposure. B: concentration-response relations determined by least-squares iterative fit of Hill equation to mean {Delta}Ppa in A, expressed as percentages of {Delta}Ppa in control lungs. In the case of ML-9 and ML-7, data obtained during the 3rd exposure were used. In the case of Y-27632 and HA-1077, data obtained during the 4th exposure were used.

 
Responses to Depolarization

PASMC. On exposure to 60 mM KCl, [Ca2+]i exhibited an initial rapid overshoot followed by a sustained elevation (Fig. 7A). The peak increase caused by depolarization was usually larger than that caused by hypoxia (Fig. 1). ML-9 and ML-7, antagonists of MLCK, caused concentration-dependent inhibition of this response (IC50 = 19.1 and 4.6 µM, respectively; Fig. 7). Similar to their lack of effect on CCE during normoxia (Fig. 2), the ROK antagonists Y-27632 (10 µM) and HA-1077 (100 µM) did not alter depolarization-induced increases in [Ca2+]i during normoxia (Fig. 7).


Figure 7
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  		Fig. 7. Effects of antagonists of MLCK (ML-9 and ML-7) and ROK (Y-27632 and HA-1077) on [Ca2+]i responses to 60 mM KCl in rat distal PASMC. A: time course of responses to KCl at different antagonist concentrations. B: average change in [Ca2+]i during 2–10 min of exposure to 60 mM KCl in A in untreated control PASMC and cells treated with MLCK and ROK antagonists. Concentrations of ROK antagonists that caused maximum inhibition of [Ca2+]i responses to hypoxia (Fig. 1) had no effect on KCl responses during normoxia. C: concentration-response relations determined by least-squares iterative fit of Hill equation to mean data in B, expressed as percentages of values in control cells.

 
To confirm that the inhibitory effect of ML-9 on the [Ca2+]i response to depolarization was due to inhibition of Ca2+ entry through VOCC, we measured Mn2+ quenching of fura 2 fluorescence in PASMC perfused with Ca2+-free KRBS containing 60 mM KCl to activate VOCC and 50 µM SKF-96365 to block CCE through SOCC (65, 66). Under these conditions, 100 µM ML-9 and 5 µM nifedipine markedly reduced the rate at which Mn2+ quenched fura 2 fluorescence (Fig. 8).


Figure 8
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  		Fig. 8. Effect of antagonists of MLCK (100 µM ML-9) and L-type voltage-operated Ca2+ channels (5 µM nifedipine) on voltage-operated Ca2+ entry, measured as quenching of fura 2 fluorescence at 360 nm (F360) by 200 µM MnCl2 in PASMC perfused with Ca2+-free KRBS containing 0.5 mM EGTA, 50 µM SKF-96365 (SKF), and 60 mM KCl. A: time course of F360 expressed as a percentage of its value at time 0, the beginning of MnCl2 exposure. B: mean quenching of fura 2 fluorescence at 10 min calculated as 100 – F360 at 10 min, where F360 is expressed as a percentage of its value at time 0.

 
Isolated lungs. Figure 9 shows representative recordings of pressor responses to increased perfusate [KCl] in untreated control lungs and lungs treated with an antagonist of MLCK (ML-9) or ROK (HA-1077). In control lungs, increases in perfusate [KCl] caused progressive increases in Ppa from 9.7 ± 0.62 mmHg at 5 mM to 33.8 ± 2.8 mmHg at 40 mM, the highest concentration achieved in all lungs of this group. Antagonists of MLCK and ROK inhibited pressor responses to KCl (Fig. 10A). Estimated IC50 values for ML-9, ML-7, Y-27632, and HA-1077 were 57.3, 79.0, 6.0, and 11.6 µM, respectively (Fig. 10B). In the case of ML-9 and ML-7, these IC50 values were 0.5–1.2 log units greater than IC50 values measured vs. [Ca2+]i responses to depolarization in PASMC (19.1 and 4.6 µM, respectively; Fig. 7B). This was not true for the ROK antagonists Y-27632 and HA-1077, which inhibited pressor responses to depolarization in isolated lungs (Fig. 10B) but had no effect on [Ca2+]i responses to depolarization in PASMC (Fig. 7B).


Figure 9
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  		Fig. 9. Representative recordings of Ppa during progressive increases in perfusate [K+] in untreated control isolated rat lungs (A) and lungs treated with an MLCK antagonist (B; ML-9) or an ROK antagonist (C; HA-1077).

 

Figure 10
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  		Fig. 10. Effects of antagonists of MLCK (ML-9 and ML-7) and ROK (Y-27632 and HA-1077) on increases in Ppa from baseline ({Delta}Ppa) caused by progressive increases in perfusate [K+] in isolated rat lungs. A: mean {Delta}Ppa at 20–40 mM K+. B: concentration-response relations determined by least-squares iterative fit of Hill equation to mean {Delta}Ppa at 40 mM K+ in A, expressed as percentages of {Delta}Ppa measured in control lungs.

 

   DISCUSSION
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 ABSTRACT
 METHODS
 RESULTS
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 REFERENCES
 
In our prior studies (66, 77), we found that HPV in isolated lungs required Ca2+ entry through SOCC and VOCC. In the present study, we confirm that HPV was blocked by antagonists of MLCK or ROK and test the possibility that this inhibition was caused by effects on Ca2+ signaling in PASMC.

In rat distal PASMC, hypoxia induced a rapid reversible increase in [Ca2+]i (Fig. 1) and enhanced CCE (Figs. 2 and 3), as previously observed in this preparation (66). Both effects were blocked by antagonists of MLCK or ROK. Moreover, antagonist IC50 values vs. [Ca2+]i responses to hypoxia were virtually identical to IC50 values vs. hypoxic enhancement of CCE (Figs. 1C and 2C). Since CCE through SOCC was essential for the [Ca2+]i response to hypoxia in PASMC (66), these results suggest that MLCK and ROK antagonists blocked [Ca2+]i responses to hypoxia by blocking CCE. This could occur if the antagonists 1) blocked Ca2+ release from the SR, thereby preventing or limiting depletion of SR Ca2+ stores, and/or 2) inhibited transduction pathways signaling activation of SOCC in response to SR Ca2+ store depletion.

To test the first possibility, we determined whether ML-9 or Y-27632 altered the effects of hypoxia on [Ca2+]i in PASMC perfused with Ca2+-free KRBS (Fig. 4). Under these conditions, any increase in [Ca2+]i must be due to release of Ca2+ from intracellular stores. Consistent with previous evidence in PASMC and pulmonary arteries indicating that hypoxia stimulates release of Ca2+ from the SR (14, 24, 49, 64, 67, 84, 85), we found that hypoxia caused a transient increase in [Ca2+]i in these cells. This increase was abolished by ML-9 or Y-27632 (Fig. 4), suggesting that ML-9 and Y-27632 inhibited [Ca2+]i responses to hypoxia (Fig. 1) by blocking hypoxia-induced release of Ca2+ from the SR, thereby preventing SOCC activation. Neither ML-9 nor Y-27632 alone altered [Ca2+]i in PASMC exposed to Ca2+-free conditions; therefore, it is unlikely that these antagonists inhibited hypoxia-induced Ca2+ release by causing prior depletion of SR Ca2+ stores (43). Rather, they must have interfered with the poorly understood mechanisms by which hypoxia activates Ca2+ release in PASMC.

With respect to the second possibility, we found that hypoxia did not alter Ca2+ release induced by CPA but, nevertheless, enhanced CPA-induced CCE (see RESULTS, Figs. 2 and 3) (66), suggesting that hypoxia facilitated transduction pathways linking store depletion to SOCC activation. Antagonists of MLCK and ROK blocked hypoxic enhancement of CPA-induced CCE, suggesting interference with this facilitation (Figs. 2 and 3). Thus MLCK and ROK antagonists appeared to block [Ca2+]i responses to hypoxia by inhibiting hypoxic release of Ca2+ from the SR and hypoxic facilitation of the transduction pathways leading to SOCC activation; however, if SR Ca2+ stores acted upon by hypoxia differed from those acted upon by CPA, as suggested by some investigators (20, 24, 25, 62), hypoxic facilitation of transduction pathways leading to SOCC activation would not necessarily be implicated by our results. More work is needed to clarify the mechanisms by which hypoxia activates CCE.

Hypoxia activated MLCK and ROK in PASMC and pulmonary arteries, leading to MLC phosphorylation and contraction that was inhibited by ML-9 or Y-27632 (33, 38, 68, 69, 83). Inhibition of HPV in isolated lungs by Y-27632 has also been reported (16, 47). Our results confirm these findings and, in addition, demonstrate inhibition of HPV in isolated lungs by ML-9, ML-7, and HA-1077 (Figs. 5 and 6). Although MLCK and ROK antagonists may have blocked HPV by inhibiting actin-myosin cross-bridge cycling and contraction, as commonly assumed, our results in PASMC (Fig. 1) suggest that they also may have acted by inhibiting Ca2+ signaling.

This deduction assumes that the behavior of our in vitro PASMC was similar to that of PASMC in vivo. In support of this assumption, acute hypoxia caused a rapid reversible increase in [Ca2+]i in our PASMC (but not in similarly treated aortic smooth muscle cells), which was half-maximal at 39 mmHg PO2 and inhibited by exposure to Ca2+-free medium or antagonists of voltage- and store-operated channels (66). Although these response characteristics were shared by HPV in isolated rat lungs (77), more direct evidence is needed to confirm that Ca2+ signaling in our PASMC reflected that of in vivo cells.

Because HPV in isolated lungs and [Ca2+]i responses to hypoxia in PASMC depended on Ca2+ influx through SOCC and VOCC (42, 66, 77), we determined the effects of MLCK and ROK antagonists on responses to activation of VOCC with high extracellular [K+]. In PASMC, ML-9 and ML-7 blocked increases in [Ca2+]i caused by high [K+] (Fig. 7), suggesting inhibition of Ca2+ entry through VOCC. To test this possibility, we measured effects of ML-9 and nifedipine on the rate at which Mn2+ quenched fura 2 fluorescence in PASMC perfused with Ca2+-free KRBS containing 60 mM KCl to activate VOCC and 50 µM SKF-96365 to block entry through SOCC and nonspecific cation channels (65, 66). Similar to nifedipine, ML-9 abolished the increased quenching caused by high [K+] (Fig. 8), suggesting that ML-9 blocked Ca2+ entry through VOCC. Thus the effects of ML-9 on the [Ca2+]i response to hypoxia (Fig. 1) may have been due in part to inhibition of VOCC, which contributed to this response in rat distal PASMC (66). In contrast, neither Y-27632 nor HA-1077 altered [Ca2+]i responses to high extracellular [K+] (Fig. 7). This result suggests that ROK was not activated in PASMC under normoxic conditions. Consistent with this possibility, neither Y-27632 nor HA-1077 altered CCE in PASMC during normoxia, whereas ML-9 caused inhibition (Fig. 2).

As expected, MLCK antagonists blocked pulmonary vasoconstrictor responses to high perfusate [K+] in isolated lungs (Figs. 9 and 10). Because of our results in PASMC (Figs. 8 and 9), this effect could be due to inhibition of the actin-myosin interaction and/or inhibition of voltage-operated Ca2+ entry. In contrast to their lack of effect on [Ca2+]i responses to high [K+] in PASMC, ROK antagonists were potent inhibitors of pulmonary pressor responses to high [K+] in isolated lungs, as previously reported (16). ROK antagonists have had similar inhibitory effects on contractile responses to high [K+] in most (18, 27, 32, 37, 59), but not all (22, 47, 63), studies of airway and vascular tissue. These effects may have occurred because ROK had been activated by paracrine mediators released from depolarized neural, endothelial, or other cells present in isolated lungs, vessels, and airways, but not our PASMC preparation. Consistent with this possibility, we found that Y-27632 blocked responses to high [K+] in intact, but not endothelium-denuded, rat distal pulmonary arteries (78).

IC50 values of MLCK and ROK antagonists vs. Ca2+ signaling during hypoxia in PASMC (Fig. 1C) were slightly greater than IC50 values vs. kinase activities of the purified enzymes measured in vitro (3, 12, 48, 55) and less than IC50 values vs. HPV in isolated lungs (Fig. 6B); however, they were comparable to concentrations found by other laboratories to inhibit smooth muscle contraction or MLC phosphorylation induced by high extracellular [K+] or [Ca2+]i in the case of ML-9 and ML-7 (23, 37, 48, 78, 80) or by G protein-linked receptor agonists or GTP{gamma}S in the case of Y-27632 and HA-1077 (10, 18, 22, 26, 37, 40, 55, 59, 63, 78). Since MLCK and ROK antagonists act by competing with ATP for its binding sites on the enzymes, these differences could be due to different concentrations of ATP and/or enzymes among preparations (3, 40, 48, 55). Moreover, in isolated lungs and smooth muscle preparations, MLCK and ROK activity may have been increased by endogenous or exogenous agonists, thereby increasing antagonist concentrations required for inhibition (26, 47, 55, 70), an effect that would not be present in purified enzyme preparations (3, 12, 48, 55). For example, the presence of an endogenous agonist might explain why HPV was slightly, but significantly, increased by 30 µM ML-9 or ML-7 during the fourth exposure of our isolated lungs to hypoxia (Fig. 6A). Since MLCK antagonists blocked Ca2+ influx and nitric oxide production in endothelial cells (74), they may have decreased endothelium-dependent vasodilation, a known modulator of HPV (21). An exogenous agonist might explain why IC50 for Y-27632 vs. HPV was 31.2 µM in our isolated lungs, which were exposed to ANG II and hypoxia, but only 0.06 µM in the lungs studied by Robertson et al. (47), which were exposed to hypoxia alone. Alternatively, this difference in IC50 could be related to the phase of HPV, since phase 2 HPV in pulmonary arteries was more sensitive to Y-27632 than phase 1 (47) and since the duration of hypoxia in our lungs was only 10–15 min compared with 40 min in the study of Robertson et al. Additional factors that could decrease antagonist potency in isolated lungs include protein binding, diffusion barriers, catabolism, and a large volume of distribution. Such considerations emphasize that comparability of IC50 values does not provide much support for the possibility that the inhibitory effects of MLCK and ROK antagonists on Ca2+ signaling in HPV were indeed due to inhibition of MLCK or ROK, rather than to nonspecific actions, such as inhibition of other kinases. Nevertheless, this possibility should be considered.

If MLCK and ROK antagonists acted by decreasing [P-MLC], we must explain how this decrease led to such diverse effects on Ca2+ signaling; i.e., MLCK and ROK antagonists blocked hypoxia-induced increases in [Ca2+]i (Fig. 1) by blocking Ca2+ release from the SR (Fig. 4), Ca2+ entry through SOCC (Figs. 2 and 3), and Ca2+ entry through VOCC (Figs. 7 and 8). One possible explanation is that antagonist-induced decreases in [P-MLC] inhibited myosin motors and/or altered the cytoskeleton in a manner that prevented necessary maintenance or rearrangement of spatial relationships among the cellular components of the hypoxic response. For example, hypoxic activation of SR Ca2+ release by cADP-ribose (13, 79) and/or short-lived reactive oxygen species released from mitochondria (75, 76) might require proximity of mitochondria to SR. SOCC activation might depend on translocation of channel proteins to the plasma membrane (5, 51) and/or assembly of channels and regulatory proteins into signaling scaffolds (4, 7, 8, 81). Maintenance of VOCC activation might require local sequestration of inactivating Ca2+ by mitochondria transported to the plasma membrane (6, 11, 15) or stimulation of integrin-linked transduction pathways by increased cytoskeletal tension (34).

Such possibilities would be consistent with accumulating evidence that the cytoskeleton plays an important role in Ca2+ signaling of smooth muscle contraction. In vascular and airway smooth muscle, antagonists of actin polymerization inhibited contractile responses (1, 9, 36) and currents through L-type VOCC (41). Depletion of focal adhesion kinase, which binds to integrins that link the cytoskeleton to the extracellular matrix and phosphorylates several cytoskeletal proteins, inhibited agonist-induced increases in [Ca2+]i, MLC phosphorylation, and contraction (60). Anti-{alpha}-integrin antibody or fibronectin, an extracellular matrix protein that binds to integrins, activated currents through L-type VOCC in vascular smooth muscle (82). This effect was inhibited by antibodies against focal adhesion kinase and the cytoskeletal proteins vinculin and paxillin. In endothelial cells, inhibition of MLCK with ML-9 blocked CCE (43, 58, 72–74). Extracted MLCK coprecipitated with actin and spectrin, a cytoskeletal protein that links to actin and plasmalemmal proteins (4, 8). CCE was blocked by stabilizing or depolymerizing the actin cytoskeleton (43), preventing interaction of spectrin with protein 4.1, which tethers spectrin to the plasma membrane and regulates spectrin-actin cross-linking (81), or blocking binding of protein 4.1 to TRPC4 protein, which is thought to be a component of SOCC (7). Thus it seems possible that myosin motors or cytoskeletal effects of actin-myosin interaction could be essential components of Ca2+ signaling in PASMC during hypoxia. Whether this is true and whether antagonists of MLCK and ROK inhibited hypoxic Ca2+ signaling by interfering with these components remain to be determined.

In summary, our results indicate that the MLCK antagonists ML-9 and ML-7 and the ROK antagonists Y-27632 and HA-1077 blocked HPV in isolated lungs and increases in [Ca2+]i induced by hypoxia in distal PASMC. The latter effect appeared to be due to inhibition of Ca2+ release from the SR, Ca2+ entry through SOCC, and Ca2+ entry through VOCC. If the behavior of our PASMC resembled that of PASMC in vivo, these results suggest that MLCK and ROK antagonists blocked HPV by inhibiting Ca2+ signaling, as well as the actin-myosin interaction.


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 ABSTRACT
 METHODS
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 GRANTS
REFERENCES
 
This work was supported by National Heart, Lung, and Blood Institute Grants HL-51912 (J. T. Sylvester), HL-75113 (J. T. Sylvester), and HL-67919 (L. A. Shimoda), an American Heart Association Scientist Development Grant (J. Wang), and National Heart, Lung, and Blood Institute Independent Scientist Award HL-079981 (J. Wang).


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. T. Sylvester, Div. of Pulmonary & Critical Care Medicine, The Johns Hopkins Asthma and Allergy Center, 5501 Hopkins Bayview Circle, Baltimore, MD 21224 (e-mail: jsylv{at}jhmi.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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