Capacitative Ca2+ entry and tyrosine kinase activation in canine pulmonary arterial smooth muscle cells

Shouzaburoh Doi, Derek S. Damron, Mayumi Horibe, and Paul A. Murray

Center for Anesthesiology Research, Division of Anesthesiology and Critical Care Medicine, The Cleveland Clinic Foundation, Cleveland, Ohio 44195


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We investigated the role of capacitative Ca2+ entry and tyrosine kinase activation in mediating phenylephrine (PE)-induced oscillations in intracellular free Ca2+ concentration ([Ca2+]i) in canine pulmonary arterial smooth muscle cells (PASMCs). [Ca2+]i was measured as the 340- to 380-nm ratio in individual fura 2-loaded PASMCs. Resting [Ca2+]i was 96 ± 4 nmol/l. PE (10 µmol/l) stimulated oscillations in [Ca2+]i, with a peak amplitude of 437 ± 22 nmol/l and a frequency of 1.01 ± 0.12/min. Thapsigargin (1 µmol/l) was used to deplete sarcoplasmic reticulum (SR) Ca2+ after extracellular Ca2+ was removed. Under these conditions, a nifedipine-insensitive, sustained increase in [Ca2+]i (140 ± 7% of control value) was observed when the extracellular Ca2+ concentration was restored; i.e., capacitative Ca2+ entry was demonstrated. Capacitative Ca2+ entry also refilled SR Ca2+ stores. Capacitative Ca2+ entry was attenuated (32 ± 3% of control value) by 50 µmol/l of SKF-96365 (a nonselective Ca2+-channel inhibitor). Tyrosine kinase inhibition with tyrphostin 23 (100 µmol/l) and genistein (100 µmol/l) also inhibited capacitative Ca2+ entry to 63 ± 12 and 85 ± 4% of control values, respectively. SKF-96365 (30 µmol/l) attenuated both the amplitude (15 ± 7% of control value) and frequency (50 ± 21% of control value) of PE-induced Ca2+ oscillations. SKF-96365 (50 µmol/l) abolished the oscillations. Tyrphostin 23 (100 µmol/l) also inhibited the amplitude (17 ± 7% of control value) and frequency (45 ± 9% of control value) of the oscillations. Genistein (30 µmol/l) had similar effects. Both SKF-96365 and tyrphostin 23 attenuated PE-induced contraction in isolated pulmonary arterial rings. These results demonstrate that capacitative Ca2+ entry is present and capable of refilling SR Ca2+ stores in canine PASMCs and may be involved in regulating PE-induced Ca2+ oscillations. A tyrosine kinase is involved in the signal transduction pathway for alpha 1-adrenoreceptor activation in PASMCs.

alpha 1-adrenoreceptor activation


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

VASOMOTOR TONE AND REACTIVITY are regulated by intracellular free Ca2+ concentration ([Ca2+]i) and myofilament sensitivity for Ca2+. Hamada et al. (13) have recently reported that alpha 1-adrenoreceptor activation with phenylephrine induces oscillations in [Ca2+]i in individual freshly dispersed and cultured pulmonary arterial smooth muscle cells (PASMCs). The maintenance of these oscillations requires sarcolemmal Ca2+ influx, is independent of voltage-gated Ca2+-channel activation, and involves the release of Ca2+ from thapsigargin-sensitive intracellular Ca2+ stores (13). However, the sarcolemmal Ca2+ influx pathway for phenylephrine-induced Ca2+ oscillations in PASMCs has not been identified.

Capacitative Ca2+ entry involves the sarcolemmal influx of Ca2+ in response to depletion of intracellular Ca2+ stores (23). This Ca2+ entry pathway does not involve voltage-gated Ca2+ channels (23). Because of the aforementioned characteristics of phenylephrine-induced Ca2+ oscillations, in the present study, we tested the hypothesis that capacitative Ca2+ entry was involved in the regulation of Ca2+ oscillations in response to alpha 1-adrenoreceptor activation in PASMCs. Because tyrosine kinase activation has been implicated in the regulation of alpha -adrenoreceptor-mediated contraction in rat pulmonary arteries (15, 26) as well as in the regulation of agonist-induced capacitative Ca2+ entry in pulmonary arteries (10) and non-PASMC types (8, 16, 24), we also tested the hypothesis that tyrosine kinase activation was involved in the signal transduction pathway for phenylephrine-induced Ca2+ oscillations.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. Pulmonary arteries were isolated from adult mongrel dogs. The technique of euthanasia was approved by the Institutional Animal Care and Use Committee. All steps were performed aseptically under general anesthesia (20 µg/kg iv of fentanyl and 30 mg/kg iv of pentobarbital sodium) with endotracheal intubation and positive-pressure ventilation. The dogs were exsanguinated by removing the mobilizable blood volume and then euthanized by administering saturated KCl (30 ml). A thoracotomy was performed via the left fifth intercostal space. The heart and lungs were removed en bloc, and the pulmonary arteries were isolated and dissected in a laminar flow hood under sterile conditions.

Cell culture of PASMCs. Primary cultures of PASMCs were obtained as previously described (13). The intralobar arteries (2- to 4-mm ID) were carefully dissected and prepared for tissue culture. Explant cultures were prepared according to the method of Campbell and Campbell (2) with minor modifications. Briefly, the endothelium and adventitia were removed together with the most superficial part of the tunica media. The media was cut into 2-mm2 pieces, explanted in 25-cm2 culture dishes nourished by DMEM-Ham's F-12 medium containing 10% fetal bovine serum and a 1% antibiotic-antimycotic mixture solution (10,000 units/ml of penicillin, 10,000 mg/ml of streptomycin, and 25 mg/ml of amphotericin B), and kept in a humidified atmosphere of 5% CO2-95% air at 37°C. PASMCs began to proliferate from explants after 7 days in culture. The cells were allowed to grow for an additional 7-10 days before being subcultured nonenzymatically to 75-cm2 culture flasks and/or 35-mm glass dishes designed for fluorescence microscopy (Bioptechs Delta T System). Cells were used for experimentation within 72 h.

Fura 2-loading procedure. PASMCs were loaded with fura 2 as previously described (13). Twenty-four hours before experimentation, the culture medium containing 10% fetal bovine serum was replaced with a serum-free medium to arrest cell growth and allow for establishment of steady-state cellular events independent of cell division. PASMCs were washed twice in loading buffer (LB), which contained (in mmol/l) 125 NaCl, 5 KCl, 1.2 MgSO4, 11 glucose, 1.8 CaCl2, and 25 HEPES plus 0.2% BSA, pH 7.40 adjusted with NaOH. PASMCs were then incubated in LB containing 2 µmol/l of fura 2-AM (Molecular Probes) at ambient temperature for 30 min. After the 30-min loading period, the cells were washed twice in LB and incubated at ambient temperature for an additional 20 min before study.

Measurement of [Ca2+]i. [Ca2+]i was measured as previously described (13). Culture dishes containing fura 2-loaded PASMCs were placed in a temperature-regulated (37°C) chamber (Bioptechs) mounted on the stage of an Olympus IX-70 inverted fluorescence microscope. Fluorescence measurements were performed on individual PASMCs in a culture monolayer with a dual-wavelength spectrofluorometer (Deltascan RFK6002, Photon Technology International) at excitation wavelengths of 340 and 380 nm and an emission wavelength of 510 nm. The volume of the chamber was 1.5 ml. The cells were superfused continuously at 1 ml/min with Krebs-Ringer buffer, which contained (in mmol/l) 125 NaCl, 5 KCl, 1.2 MgSO4, 11 glucose, 2.5 CaCl2, and 25 HEPES, pH 7.40 adjusted with NaOH. The temperature of all solutions was maintained at 37°C in a water bath. Solution changes were accomplished rapidly by aspirating the buffer in the dish and transiently increasing the flow rate to 10 ml/min. Just before data acquisition, background fluorescence (i.e., fluorescence between cells) was measured and subtracted automatically from the subsequent experimental measurements. Fura 2 fluorescence signals (340 nm, 380 nm, and 340- to 380-nm ratio) originating from individual PASMCs were continuously monitored at a sampling frequency of 25 Hz and collected with a software package from Photon Technology International (Felix).

Fura 2 titration. Estimations of [Ca2+]i were achieved by comparing the cellular fluorescence ratio with fluorescence ratios acquired with fura 2 (free acid) in buffers containing known Ca2+ concentrations. [Ca2+]i was calculated as described by Grynkiewicz et al. (12).

Organ chamber experiments. Canine intralobar pulmonary arteries (2- to 4-mm ID) were isolated and prepared as previously described (28). The arteries were cut into 0.5-cm-wide rings, and the endothelium was intentionally removed by inserting forceps tips into the vessel lumen and rolling the rings over damp filter paper. Endothelial denudation was later confirmed by the absence of relaxation to acetylcholine (1 µmol/l). The rings were suspended horizontally between two stainless steel stirrups in organ chambers filled with 25 ml of modified Krebs-Ringer bicarbonate solution (37°C) gassed with 95% O2-5% CO2. One of the stirrups was anchored, and the other was connected to a strain gauge (Grass model FT03) for measurement of isometric tension. The rings from the same relative anatomic locations in the right and left lungs were used as paired rings. The rings were stretched at 10-min intervals in increments of 0.5 g to reach optimal resting tone (5 g). The contractile response to 60 mmol/l of KCl was measured. After washout of KCl from the organ chambers and the return of isometric tension to prestimulation values, concentration-effect curves for phenylephrine were performed in untreated rings and rings pretreated with either SKF-96365 (50 µmol/l) or tyrphostin 23 (100 µmol/l). All rings were pretreated with propranolol (5 µmol/l; incubated for 30 min) to inhibit the beta -agonist effects of phenylephrine.

Drug preparation. Phenylephrine and propranolol (Sigma) were prepared as 10 and 5 mmol/l stock solutions, respectively, in distilled water. Aliquots of the stock solutions were diluted 1:1,000 in Krebs-Ringer buffer to achieve final concentrations of 10 and 5 µmol/l, respectively. Acetylcholine (Sigma) was dissolved in distilled water to achieve a final organ bath concentration of 1 µmol/l. SKF-96365, tyrphostin 23, tyrphostin A, genistein, cyclopiazonic acid (CPA), and ML-7 (Calbiochem) as well as thapsigargin, ionomycin, and nifedipine (Sigma) were dissolved in DMSO as stock solutions. Aliquots of each stock solution were diluted 1:1,000 in Krebs-Ringer buffer to achieve final concentrations. Similar dilutions of DMSO in Krebs-Ringer buffer had no effect on [Ca2+]i (13).

Data analysis. Peak and sustained increases in [Ca2+]i were measured in individual PASMCs when the superfusion solution was switched from a Ca2+-free solution to a solution containing 2.2 mmol/l of Ca2+. Peak and sustained ratio values were averaged before and after each intervention and are expressed as a percentage of the control value. The amplitude and frequency of phenylephrine-induced increases in [Ca2+]i were measured in individual PASMCs. The amplitude was calculated by averaging the peak ratio value for four to five oscillations measured before (control) and after each intervention. Changes in the amplitude of the 340- to 380-nm fluorescence ratio in response to an intervention are expressed as a percentage of the control value. The frequency of oscillations was calculated by averaging the time interval between the oscillation peaks and is reported as the number of oscillations observed per minute. Changes in the frequency of oscillations are also expressed as a percentage of control values. Phenylephrine-induced increases in isometric tension are expressed as the percentage of the contractile response to 60 mmol/l of KCl. The results are presented as means ± SE. Statistical analysis was performed with repeated-measures ANOVA followed by Bonferroni-Dunn post hoc testing. Differences were considered significant at P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Efficacy of thapsigargin to deplete sarcoplasmic reticulum Ca2+ stores. Capacitative Ca2+ entry is triggered by the depletion of intracellular Ca2+ stores. Thapsigargin increases [Ca2+]i in a variety of cell types via irreversible inhibition of sarcoplasmic reticulum (SR) Ca2+-ATPase (32). The membrane permeabilizing agent ionomycin (5 µmol/l) was used to assess the efficacy of thapsigargin (1 µmol/l) to deplete SR Ca2+ stores. In the absence of extracellular Ca2+, an increase in the fluorescence signal in response to ionomycin should reflect the release of Ca2+ from intracellular stores. Ionomycin stimulated a large [Ca2+]i transient, reaching a peak fluorescence ratio of 8.8 ± 0.7 (Fig. 1). After pretreatment with thapsigargin (1 µmol/l), ionomycin again stimulated an increase in [Ca2+]i, but the peak fluorescence ratio reached only 2.2 ± 0.2. Figures 1-10 each show a representative trace depicting the effect of each intervention on [Ca2+]i and the summarized data.


View larger version (12K):
[in this window]
[in a new window]
 
Fig. 1.   A: representative trace depicting effect of ionomycin, a membrane-permeabilizing agent, on baseline intracellular Ca2+ concentration ([Ca2+]i) with and without thapsigargin, a Ca2+-ATPase inhibitor, pretreatment in absence of extracellular Ca2+ (Ca2+-free buffer plus 2 mmol/l of EGTA). Ionomycin (5 µmol/l) and thapsigargin (1 µmol/l) were added to superfusion buffer (arrows). B: summarized data depicting inhibitory effect of thapsigargin on increase in [Ca2+]i induced by ionomycin in absence of extracellular Ca2+ (n = 4 cells). 340/380 Fluorescence Ratio, 340- to 380-nm fluorescence ratio. * P < 0.05.

Capacitative Ca2+ entry after depletion of SR Ca2+ stores. To identify the presence of capacitative Ca2+ entry in canine PASMCs, SR Ca2+ stores were depleted with thapsigargin (1 µmol/l) in the absence of extracellular Ca2+ (Fig. 2). Once the baseline fluorescence signal had stabilized, the extracellular Ca2+ concentration ([Ca2+]o) was restored (2.2 mmol/l) in the continued presence of thapsigargin. Restoring [Ca2+]o resulted in a rapid peak increase (197 ± 11% of the control value) and a sustained increase (140 ± 7% of the control value) in [Ca2+]i. The sustained increase in [Ca2+]i returned to baseline when extracellular Ca2+ was removed.


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 2.   A: representative trace depicting capacitative Ca2+ entry after depletion of sarcoplasmic reticulum (SR) Ca2+ stores with thapsigargin. In absence of extracellular Ca2+ (Ca2+-free buffer plus 2 mmol/l of EGTA), thapsigargin stimulated a transient increase in [Ca2+]i by releasing Ca2+ from SR Ca2+ stores. After [Ca2+]i had returned to baseline levels, Ca2+ was added back to buffer, which caused sustained capacitative Ca2+ entry. [Ca2+]i returned to baseline levels after removal of extracellular Ca2+. B: summarized data depicting peak and sustained increases in [Ca2+]i due to capacitative Ca2+ entry (n = 12 cells). * P < 0.05 compared with baseline.

Capacitative Ca2+ entry refills SR Ca2+ stores. CPA (10 µmol/l), a reversible SR Ca2+-ATPase inhibitor, was used to reversibly deplete SR Ca2+ stores in the absence of extracellular Ca2+ (19). CPA stimulated an increase in [Ca2+]i similar to that induced by thapsigargin (Fig. 3). After washout of CPA, [Ca2+]o was restored (2.2 mmol/l), and a transient increase in [Ca2+]i was observed. This effect was not sustained because Ca2+ was taken up by the SR. To confirm this, in the absence of extracellular Ca2+, readdition of CPA stimulated an increase in [Ca2+]i similar in magnitude to that induced by CPA the first time. This demonstrates that SR Ca2+ stores were refilled when Ca2+ was restored to the extracellular space.


View larger version (21K):
[in this window]
[in a new window]
 
Fig. 3.   A: representative trace depicting role of capacitative Ca2+ entry in refilling SR Ca2+ stores. In absence of extracellular Ca2+, cyclopiazonic acid (CPA) stimulated a transient increase in [Ca2+]i by releasing Ca2+ from SR Ca2+ stores. After [Ca2+]i had returned to baseline levels, CPA was washed out and Ca2+ was added back to buffer. A transient increase in [Ca2+]i, reflecting capacitative Ca2+ entry, was observed. This effect was not sustained because Ca2+ was taken up by SR. To confirm this, readdition of CPA stimulated an increase in [Ca2+]i similar in magnitude to that induced by CPA the first time. B: summarized data showing that CPA induced capacitative Ca2+ entry and refilling of SR Ca2+ stores (n = 7 cells).

Reproducibility of capacitative Ca2+ entry. We examined the reproducibility of inducing capacitative Ca2+ entry in the same PASMCs. After thapsigargin (1 µmol/l) pretreatment to deplete SR Ca2+ stores, [Ca2+]o was sequentially restored (2.2 mmol/l) and removed (0 mmol/l) three consecutive times to trigger capacitative Ca2+ entry (Fig 4). Thapsigargin was present throughout the entire protocol. There were no significant differences in the peak or sustained increases in [Ca2+]i when [Ca2+]o was restored three consecutive times.


View larger version (33K):
[in this window]
[in a new window]
 
Fig. 4.   A: representative trace showing that in absence of extracellular Ca2+ (Ca2+-free buffer plus 2 mmol/l of EGTA), thapsigargin stimulated a transient increase in [Ca2+]i by releasing Ca2+ from SR Ca2+ stores. After depletion of SR Ca2+ stores with thapsigargin, extracellular Ca2+ (2.2 mmol/l) was sequentially added and removed 3 times. B: summarized data showing reproducibility of peak and sustained increases in [Ca2+]i due to capacitative Ca2+ entry with repeated challenges (n = 4 cells).

Effect of nifedipine on capacitative Ca2+ entry. Nifedipine (10 µmol/l), a voltage-dependent Ca2+-channel blocker, was applied just before restoring [Ca2+]o the second time (Fig. 5). Nifedipine had no effect on the peak (101 ± 5% of the control value) or sustained (97 ± 3% of the control value) increases in [Ca2+]i due to capacitative Ca2+ entry.


View larger version (28K):
[in this window]
[in a new window]
 
Fig. 5.   A: representative trace depicting effect of nifedipine on capacitative Ca2+ entry. After depletion of SR Ca2+ stores with thapsigargin, capacitative Ca2+ entry was compared in absence and presence of nifedipine, which was added to superfusion buffer. B: summarized data showing that nifedipine (NIF) had no effect on peak and sustained increases in [Ca2+]i due to capacitative Ca2+ entry (n = 3 cells).

Effect of SKF-96365 on capacitative Ca2+ entry. SKF-96365 is a nonselective Ca2+-channel blocker that has been utilized by a number of investigators (4, 17, 27, 33) to inhibit capacitative Ca2+ entry. SKF-96365 (50 µmol/l) was applied just before [Ca2+]o was restored the second time (Fig. 6). SKF-96365 attenuated both the peak (25 ± 4% of the control value) and sustained (32 ± 3% of the control value) increases in [Ca2+]i due to capacitative Ca2+ entry.


View larger version (25K):
[in this window]
[in a new window]
 
Fig. 6.   A: Representative trace depicting effect of SKF-96365 on capacitative Ca2+ entry. After depletion of SR Ca2+ stores with thapsigargin, capacitative Ca2+ entry was compared in absence and presence of SKF-96365, which was added to superfusion buffer. B: summarized data showing inhibitory effect of 50 µmol/l of SKF-96365 [SKF(50)] on peak and sustained increases in [Ca2+]i due to capacitative Ca2+ entry (n = 7 cells). * P < 0.05.

Effects of tyrphostin 23 and genistein on capacitative Ca2+ entry. Because of interactions between the tyrosine kinase and myosin light chain kinase pathways, experiments were performed in PASMCs pretreated with the myosin light chain kinase inhibitor ML-7 (10 µmol/l). ML-7 alone had no effect on capacitative Ca2+ entry (Fig. 7). Tyrphostin 23 (100 µmol/l) and genistein (100 µmol/l) were used to inhibit tyrosine kinases. Tyrphostin 23 attenuated the peak (66 ± 9% of the control value) and sustained (63 ± 12% of the control value) increases in [Ca2+]i due to capacitative Ca2+ entry. Genistein also reduced capacitative Ca2+ entry. Tyrphostin A (100 µmol/l), an inactive analog of tyrphostin 23, had no effect on capacitative Ca2+ entry.


View larger version (37K):
[in this window]
[in a new window]
 
Fig. 7.   A: representative trace depicting effects of ML-7 (myosin light chain kinase inhibitor), tyrphostin A (Tyr A; inactive analog of tyrphostin 23), and tyrphostin 23 (Tyr 23; tyrosine kinase inhibitor) on capacitative Ca2+ entry. After depletion of SR Ca2+ stores with thapsigargin, capacitative Ca2+ entry was compared in absence and presence of ML-7, Tyr A (100 µmol/l), Tyr 23 (100 µmol/l), or genistein (100 µmol/l), which were added to superfusion buffer. B: summarized data showing that ML-7 and Tyr A had no effect on capacitative Ca2+ entry. In ML-7-pretreated cells, Tyr 23 and genistein (Gen) inhibited peak and sustained increases in [Ca2+]i due to capacitative Ca2+ entry (n = 7 cells). * P < 0.05 compared with ML-7 and Tyr A.

Effect of alpha 1-adrenoreceptor stimulation with phenylephrine on [Ca2+]i in PASMCs. Resting values of [Ca2+]i were 96 ± 4 nmol/l (ratio 1.00 ± 0.05). PASMCs were pretreated with the beta -adrenoreceptor antagonist propranolol (5 µmol/l) to eliminate any beta -agonist effect of phenylephrine. As Hamada et al. (13) have previously demonstrated, continuous superfusion of phenylephrine (10 µmol/l) stimulated repetitive [Ca2+]i oscillations at a frequency of 1.01 ± 0.12 transients/min for >30 min (n = 14 cells). The [Ca2+]i reached an average peak value of 437 ± 22 nmol/l (ratio 4.25 ± 0.49).

Effect of SKF-96365 on phenylephrine-induced [Ca2+]i oscillations. After initiation of phenylephrine-induced [Ca2+]i oscillations, SKF-96365 was added to the superfusion buffer (Fig. 8). SKF-96365 (30 µmol/l) attenuated both the amplitude (15 ± 7% of the control value) and frequency (50 ± 21% of the control value) of the oscillations. A higher dose of SKF-96365 (50 µmol/l) completely abolished the oscillations. The oscillations immediately returned when SKF-96365 was washed out.


View larger version (36K):
[in this window]
[in a new window]
 
Fig. 8.   A: representative trace depicting effect of SKF-96365, a nonselective Ca2+-channel blocker, on phenylephrine (PE)-induced [Ca2+]i oscillations. SKF-96365 was added to superfusion buffer as indicated. B: summarized data depicting dose-dependent inhibitory effect of 30 µmol/l of SKF-96365 [SKF(30)] and SKF(50) on amplitude and frequency of PE-induced [Ca2+]i oscillations (n = 5 cells). * P < 0.05 compared with control.

Effects of tyrphostin 23 and genistein on phenylephrine-induced [Ca2+]i oscillations. Tyrphostin 23 (100 µmol/l) was added to the superfusate after induction of [Ca2+]i oscillations by phenylephrine (Fig. 9). Tyrphostin attenuated both the amplitude (17 ± 7% of the control value) and frequency (45 ± 9% of the control value) of the oscillations. The oscillations returned immediately when tyrphostin was washed out. ML-7 (10 µmol/l) had no effect on the [Ca2+]i oscillations (Fig. 10). In ML-7-pretreated PASMCs, genistein (30 µmol/l) attenuated both the amplitude (37 ± 6% of the control value) and frequency (81 ± 7% of the control value) of the oscillations (Fig. 10). A higher dose of genistein (100 µmol/l) abolished the oscillations.


View larger version (28K):
[in this window]
[in a new window]
 
Fig. 9.   A: representative trace depicting effect of Tyr 23, an inhibitor of tyrosine kinases, on PE-induced [Ca2+]i oscillations. Tyr 23 was added to superfusion buffer. B: summarized data depicting inhibitory effect of 100 µmol/l of Tyr [TYR(100)] on amplitude and frequency of PE-induced [Ca2+]i oscillations (n = 5 cells). * P < 0.05.



View larger version (31K):
[in this window]
[in a new window]
 
Fig. 10.   A: representative trace depicting effect of Gen, an inhibitor of tyrosine kinases, on PE (10 µmol/l)-induced [Ca2+]i oscillations. Gen was added to superfusion buffer as indicated. These experiments were performed in presence of ML-7. B: summarized data depicting inhibitory effect of 30 [Gen(30)] and 50 [Gen(50)] µmol/l of Gen on amplitude and frequency of PE-induced [Ca2+]i oscillations (n = 5 cells). * P < 0.05 compared with ML-7.

Effect of SKF-96365 and tyrphostin 23 on phenylephrine-induced contractions. Phenylephrine caused dose-dependent increases in isometric tension in isolated pulmonary arterial rings (Fig. 11). Both SKF-96365 (50 µmol/l) and tyrphostin 23 (100 µmol/l) reduced the maximum contractile response to phenylephrine.


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 11.   Summarized data depicting effect of SKF-96365 (A) and Tyr 23 (B) on PE dose-response relationship in isolated pulmonary arterial rings. Results are percentage of maximal response to 60 mM KCl. SKF-96365 (50 µmol/l) and Tyr 23 (100 µmol/l) attenuated PE-induced contraction (n = 5 dogs). * P < 0.05.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The goal of this study was to further elucidate the cellular mechanisms that regulate phenylephrine-induced oscillations in [Ca2+]i in PASMCs. The novel findings of this study are that capacitative Ca2+ entry exists in canine PASMCs and serves to refill the SR Ca2+ pool, which, in turn, maintains the oscillations in [Ca2+]i induced by alpha 1-adrenoreceptor activation. In addition, a tyrosine kinase is involved in the signal transduction pathway for capacitative Ca2+ entry and the maintenance of phenylephrine-induced Ca2+ oscillations as well as in the contractile response to phenylephrine.

SR Ca2+ stores in PASMCs. In the absence of extracellular Ca2+, the permeabilizing agent ionomycin causes a large transient increase in [Ca2+]i due to the release of Ca2+ from multiple intracellular Ca2+ storage sites {inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] sensitive, ryanodine and/or caffeine sensitive, or mitochondrial}. Thapsigargin, an irreversible SR Ca2+-ATPase inhibitor, is commonly used to specifically deplete Ins(1,4,5)P3-sensitive SR Ca2+ pools in vascular smooth muscle (35). Compared with ionomycin, the peak increase in [Ca2+]i induced by thapsigargin is smaller, but the duration of the increase in [Ca2+]i is more prolonged. This reflects the slow leak of Ca2+ from the SR in a Ca2+-free solution (Fig. 1). Thapsigargin pretreatment reduced the ionomycin-induced increase in [Ca2+]i by >75%. These results suggest that Ins(1,4,5)P3-sensitive Ca2+ stores are the predominant Ca2+ storage site in PASMCs and that thapsigargin significantly diminishes the content of this SR Ca2+ store. The small increase in [Ca2+]i induced by ionomycin after thapsigargin pretreatment could reflect mitochondrial Ca2+ stores and/or ryanodine-sensitive SR Ca2+ stores (37). However, Hamada et al. (13) previously demonstrated that ryanodine does not alter ionomycin-induced increases in [Ca2+]i, which suggests that a small mitochondrial pool of Ca2+ may be present in canine PASMCs.

Capacitative Ca2+ entry in PASMCs. After depletion of SR Ca2+ stores with thapsigargin, a transient peak increase followed by a sustained increase in [Ca2+]i was observed when Ca2+ (2.2 mmol/l) was restored to the buffer. These data suggest the presence of a capacitative Ca2+ entry pathway in canine PASMCs. Capacitative Ca2+ entry has previously been observed in systemic vascular smooth muscle (22, 35). We are aware of only one report that has demonstrated capacitative Ca2+ entry in PASMCs, and that result was not the major focus of that study (36). In our study, the sustained increase in [Ca2+]i induced by restoring [Ca2+]o is likely due to the ability of thapsigargin to irreversibly inhibit the SR Ca2+-ATPase. This would result in persistent capacitative Ca2+ entry due to the inability of the SR to sequester Ca2+, causing a sustained increase in [Ca2+]i. This protocol allowed us to repeatedly induce capacitative Ca2+ entry simply by restoring [Ca2+]o.

Capacitative Ca2+ entry refills SR Ca2+ stores in PASMCs. Unlike thapsigargin, CPA is a reversible inhibitor of SR Ca2+-ATPase, resulting in depletion of Ins(1,4,5)P3-sensitive SR Ca2+ stores (19). In the absence of extracellular Ca2+, CPA triggered an increase in [Ca2+]i similar to that observed with thapsigargin. However, when CPA was washed out and [Ca2+]o was restored, a rapid transient increase in [Ca2+]i was observed, with no sustained increase in [Ca2+]i, implying a refilling of SR Ca2+ stores and termination of capacitative Ca2+ entry. A second challenge with CPA in the absence of extracellular Ca2+ confirmed that the Ins(1,4,5)P3-sensitive SR Ca2+ store had been refilled by capacitative Ca2+ entry because the increase in [Ca2+]i induced by CPA was similar to the first challenge. Electrophysiological evidence suggests that capacitative Ca2+ entry is necessary for SR Ca2+ store replenishment in smooth muscle (34). Our results provide the first evidence that capacitative Ca2+ entry can refill Ins(1,4,5)P3-sensitive SR Ca2+ stores in PASMCs.

Effects of SKF-96365 and nifedipine on capacitative Ca2+ entry. It is well known that capacitative Ca2+ entry is insensitive to classic inhibitors of voltage-gated Ca2+ channels but is blocked by imidazole derivatives such as SKF-96365 (7). SKF-96365 has been utilized to block capacitative Ca2+ entry after depletion of SR Ca2+ stores in a variety of cell types (4, 17, 27, 33). In our study, SKF-96365 markedly attenuated both the peak and sustained increases in [Ca2+]i due to capacitative Ca2+ entry. Nifedipine had no effect on capacitative Ca2+ entry. These results are consistent with the concept that capacitative Ca2+ entry is insensitive to voltage-gated Ca2+-channel inhibitors. Moreover, because this protocol did not involve agonist-induced receptor activation, the inhibitory effect of SKF-96365 is likely due to inhibition of store-operated Ca2+ channels.

Effects of tyrphostin 23 and genistein on capacitative Ca2+ entry. Several mechanisms have been proposed to explain the coupling between the Ca2+ content of SR stores and capacitative Ca2+ entry. It has been proposed that the SR might possess protein kinases or phosphatases capable of altering the phosphorylation state of ion channels (14). Alternatively, depletion of intracellular Ca2+ stores may trigger tyrosine kinase activation. Depletion of intracellular Ca2+ stores increased tyrosine phosphorylation in human platelets (25). Inhibition of tyrosine kinases decreased capacitative Ca2+ entry in rat pancreatic acinar cells (38), human lymphocytes (31), platelets (24), fibroblasts (16), and umbilical vein endothelial cells (9). In the present study, tyrphostin 23 and genistein attenuated both the peak and sustained increases in [Ca2+]i due to capacitative Ca2+ entry. Therefore, it appears that tyrosine kinase activation plays a role in regulating capacitative Ca2+ entry in PASMCs.

SKF-96365 inhibits phenylephrine-induced oscillations in [Ca2+]i. Hamada et al. (13) previously demonstrated that the maintenance of phenylephrine-induced Ca2+ oscillations required the presence of extracellular Ca2+ as well as the release of Ca2+ from thapsigargin-sensitive intracellular stores, whereas the influx of Ca2+ via voltage-gated Ca2+ channels was not involved. This suggests that an alternative Ca2+ influx pathway is required for the maintenance of [Ca2+]i oscillations. We hypothesized that this influx pathway involved capacitative Ca2+ entry. SKF-96365 has been shown to inhibit Ca2+ influx induced by G protein-coupled receptor activation with thrombin (21), histamine (11), bradykinin (7, 27), and endothelin-1 (3). In the rat spleen, phenylephrine-induced contraction was reduced by SKF-96365 but not by nifedipine (1), suggesting a role for capacitative Ca2+ entry in alpha 1-adrenoreceptor-activated contraction. In our study, SKF-96365 attenuated or abolished both the amplitude and frequency of phenylephrine-induced [Ca2+]i oscillations. SKF-96365 also attenuated phenylephrine-induced contraction. Taken together with our observation that SKF-96365 inhibited capacitative Ca2+ entry, our results suggest that capacitative Ca2+ entry may be involved in maintaining phenylephrine-induced Ca2+ oscillations. Although not definitive, our results are also consistent with the possibility that capacitative Ca2+ entry is involved in phenylephrine-induced contraction.

Tyrphostin 23 and genistein inhibit phenylephrine-induced oscillations in [Ca2+]i. Although tyrosine kinase activation is generally associated with cell growth, proliferation, and differentiation (30), recent evidence suggests that tyrosine kinases are involved in phenylephrine-induced contractions in the carotid (6) and mesenteric (18) arteries, pulmonary artery (15, 26), and rat spleen (1). As noted earlier, tyrosine kinase inhibitors attenuated capacitative Ca2+ entry in a variety of cell types (16, 18, 24, 38). Genistein, another tyrosine kinase inhibitor, has been shown to inhibit the peak as well as the sustained increase in [Ca2+]i induced by phenylephrine in the canine femoral artery (5, 29). Moreover, tyrosine kinase inhibitors attenuated agonist-stimulated Ins(1,4,5)P3 production by inhibiting phosphorylation of phospholipase C-gamma (20). These results indicate that tyrosine kinases can regulate multiple points in the signal transduction pathway for alpha -adrenoreceptor activation. In our study, tyrphostin 23 and genistein attenuated the oscillations in [Ca2+]i induced by phenylephrine. Tyrphostin 23 also attenuated the contractile response to phenylephrine in pulmonary arterial rings. These data suggest that a tyrosine kinase is involved in alpha 1-adrenoreceptor-mediated Ca2+ signaling in canine PASMCs, perhaps by regulating Ins(1,4,5)P3 production and/or binding, SR Ca2+ dynamics, or capacitative Ca2+ entry.

Summary. The major finding of our study is that capacitative Ca2+ entry is present, refills SR Ca2+ stores, and is likely involved in [Ca2+]i oscillations induced by alpha 1-adrenoreceptor activation in PASMCs. In addition, a tyrosine kinase is involved in the signal transduction pathway for capacitative Ca2+ entry and phenylephrine-induced [Ca2+]i oscillations in PASMCs and phenylephrine-induced contraction in pulmonary arterial rings.


    ACKNOWLEDGEMENTS

We gratefully acknowledge the technical support of Cindy Shumaker. We would also like to thank Cassandra Talerico for outstanding work in preparing the manuscript.


    FOOTNOTES

This study was supported by National Heart, Lung, and Blood Institute Grants HL-38291 and HL-40361.

S. Doi was also supported by Dr. Jun-Ichi Yata (Department of Pediatrics, Tokyo Medical and Dental University, Tokyo, Japan).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: P. A. Murray, Center for Anesthesiology Research, The Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195 (E-mail: murrayp{at}ccf.org).

Received 5 January 1999; accepted in final form 27 August 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Burt, R. P., C. R. Chapple, and I. Marshall. The role of capacitative Ca2+ influx in the alpha 1B-adrenoreceptor-mediated contraction to phenylephrine of the rat spleen. Br. J. Pharmacol. 116: 2327-2333, 1995[Abstract].

2.   Campbell, J. H., and G. R. Campbell. Culture techniques and their applications to studies of vascular smooth muscle. Clin. Sci. (Colch.) 85: 501-513, 1993[ISI][Medline].

3.   Chan, J., and D. A. Greenberg. SK&F 96365, a receptor-mediated calcium entry inhibitor, inhibits calcium responses to endothelin-1 in NG108-15 cells. Biochem. Biophys. Res. Commun. 177: 1141-1146, 1991[ISI][Medline].

4.   Demaurex, N., D. P. Lew, and K.-H. Krause. Cyclopiazonic acid depletes intracellular Ca2+ stores and activates an influx pathway for divalent cations in HL-60 cells. J. Biol. Chem. 267: 2318-2324, 1992[Abstract/Free Full Text].

5.   Di Salvo, J., G. Pfitzer, and L. A. Semenchuk. Protein tyrosine phosphorylation, cellular Ca2+, and Ca2+ sensitivity for contraction of smooth muscle. Can. J. Physiol. Pharmacol. 72: 1434-1439, 1994[ISI][Medline].

6.   Di Salvo, J., A. Steusloff, L. Semenchuk, S. Satoh, K. Kolquist, and G. Pfitzer. Tyrosine kinase inhibitors suppress agonist-induced contraction in smooth muscle. Biochem. Biophys. Res. Commun. 190: 968-974, 1993[ISI][Medline].

7.   Fasolato, C., P. Pizzo, and T. Pozzan. Receptor-mediated calcium influx in PC12 cells. J. Biol. Chem. 265: 20351-20355, 1990[Abstract/Free Full Text].

8.   Fleming, I., and R. Busse. Tyrosine phosphorylation and bradykinin-induced signaling in endothelial cells. Am. J. Cardiol. 80: 102A-109A, 1997[Medline].

9.   Fleming, I., B. Fissthaler, and R. Busse. Calcium signaling in endothelial cells involves activation of tyrosine kinases and leads to activation of mitogen-activated protein kinases. Circ. Res. 76: 522-529, 1995[Abstract/Free Full Text].

10.   Gonzalez De La Fuente, P., J.-P. Savineau, and R. Marthan. Control of pulmonary vascular smooth muscle tone by sarcoplasmic reticulum Ca2+ pump blockers: thapsigargin and cyclopiazonic acid. Eur. J. Physiol. 429: 617-624, 1995[ISI][Medline].

11.   Graier, W. F., K. Groshner, K. Schmidt, and W. R. Kukovetz. SK&F 96365 inhibits histamine-induced formation of endothelium-derived relaxing factor in human endothelial cells. Biochem. Biophys. Res. Commun. 186: 1539-1545, 1992[ISI][Medline].

12.   Grynkiewicz, G., M. Poenie, and R. Y. Tsien. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J. Biol. Chem. 260: 3440-3450, 1985[Abstract].

13.   Hamada, H., D. S. Damron, S. J. Hong, D. R. Van Wagoner, and P. A. Murray. Phenylephrine-induced Ca2+ oscillations in canine pulmonary artery smooth muscle cells. Circ. Res. 81: 812-823, 1997[Abstract/Free Full Text].

14.   Hoth, M., and R. Penner. Calcium release-activated calcium current in rat mast cells. J. Physiol. (Lond.) 465: 359-386, 1993[Abstract].

15.   Jin, N., R. A. Siddiqui, D. English, and R. A. Rhoades. Communication between tyrosine kinase pathway and myosin light chain kinase pathway in smooth muscle. Am. J. Physiol. Heart Circ. Physiol. 271: H1348-H1355, 1996[Abstract/Free Full Text].

16.   Lee, K.-M., K. Toscas, and M. L. Villereal. Inhibition of bradykinin- and thapsigargin-induced Ca2+ entry by tyrosine kinase inhibitors. J. Biol. Chem. 268: 9945-9948, 1993[Abstract/Free Full Text].

17.   Leung, Y.-M., C.-Y. Kwan, and T.-T. Loh. Dual effect of SK&F 96365 in human leukemic HL-60 cells. Inhibition of calcium entry and activation of a novel cation influx pathway. Biochem. Pharmacol. 51: 605-612, 1996[ISI][Medline].

18.   Low, A. M. Role of tyrosine kinase on Ca2+ entry and refilling of agonist-sensitive Ca2+ stores in vascular smooth muscles. Can. J. Physiol. Pharmacol. 74: 298-304, 1996[ISI][Medline].

19.   Low, A. M., C. Y. Kwan, and E. E. Daniel. Evidence for two types of internal Ca2+ stores in canine mesenteric artery with different refilling mechanisms. Am. J. Physiol. Heart Circ. Physiol. 262: H31-H37, 1992[Abstract/Free Full Text].

20.   Marrero, M. B., W. G. Paxton, J. L. Duff, B. C. Berk, and K. E. Bernstein. Angiotensin II stimulates tyrosine phosphorylation of phospholipase C-gamma1 in vascular smooth muscle cells: nicardipine-sensitive and -insensitive pathways. J. Biol. Chem. 269: 10935-10939, 1994[Abstract/Free Full Text].

21.   Merritt, J. E., W. P. Armstrong, C. D. Benham, T. J. Hallam, R. Jacob, A. Jaxa-Chamiec, B. K. Leigh, S. A. McCarthy, K. E. Moores, and T. J. Rink. SK&F 96365, a novel inhibitor of receptor-mediated calcium entry. Biochem. J. 271: 515-522, 1990[ISI][Medline].

22.   Pacaud, P., G. Loirand, G. Grigoire, C. Mironneau, and J. Mironneau. Noradrenaline-activated heparin-sensitive Ca2+ entry after depletion of intracellular Ca2+ store in portal vein smooth muscle cells. J. Biol. Chem. 268: 3866-3872, 1993[Abstract/Free Full Text].

23.   Putney, J. W., Jr. A model for receptor-regulated calcium entry. Cell Calcium 7: 1-12, 1986[ISI][Medline].

24.   Sargeant, P., R. W. Farndale, and S. O. Sage. ADP- and thapsigargin-evoked Ca2+ entry and protein-tyrosine phosphorylation are inhibited by the tyrosine kinase inhibitors genistein and methyl-2,5-dihydroxycinnamate in fura-2-loaded human platelets. J. Biol. Chem. 268: 18151-18156, 1993[Abstract/Free Full Text].

25.   Sargeant, P., R. W. Farndale, and S. Sage. Calcium store depletion in dimethyl bapta-loaded human platelets increases protein tyrosine phosphorylation in the absence of a rise in cytosolic calcium. Exp. Physiol. 79: 269-272, 1994[Abstract].

26.   Savineau, J. P., P. Gonzalez De La Fuente, and R. Marthan. Effect of modulators of tyrosine kinase activity on agonist-induced contraction in the rat pulmonary vascular smooth muscle. Pulm. Pharmacol. 9: 189-195, 1996[ISI][Medline].

27.   Schilling, W. P., O. A. Cabello, and L. Rajan. Depletion of the inositol 1,4,5-trisphosphate-sensitive intracellular Ca2+ store in vascular endothelial cells activates the agonist-sensitive Ca2+-influx pathway. Biochem. J. 284: 521-530, 1992[ISI][Medline].

28.   Seki, S., M. Horibe, and P. A. Murray. Halothane attenuates endothelium-dependent pulmonary vasorelaxant response to the ATP-sensitive K+ channel agonist, lemakalim. Anesthesiology 87: 625-634, 1997[ISI][Medline].

29.   Semenchuk, L. A., and J. Di Salvo. Receptor-activated increases in intracellular calcium and protein tyrosine phosphorylation in vascular smooth muscle cells. FEBS Lett. 370: 127-130, 1995[ISI][Medline].

30.   Shimokado, K., T. Yokota, C. Kosaka, K. Zen, T. Sasuguri, J. Masuda, and K. Ogita. Protein tyrosine kinase inhibitors inhibit both proliferation and chemotaxis of vascular smooth muscle cells. Ann. NY Acad. Sci. 748: 171-175, 1995[ISI][Medline].

31.   Tepel, M., S. Kuhnapfel, G. Theilmeier, C. Teupe, R. Schlotmann, and W. Zidek. Filling state of intracellular Ca2+ pools triggers trans plasma membrane Na+ and Ca2+ influx by a tyrosine kinase-dependent pathway. J. Biol. Chem. 269: 26239-26242, 1994[Abstract/Free Full Text].

32.   Thastrup, O., P. J. Cullen, B. K. Drbak, M. R. Hanley, and A. P. Dawson. Thapsigargin, a tumor promoter, discharges intracellular Ca2+ stores by specific inhibition of the endoplasmic reticulum Ca2+-ATPase. Proc. Natl. Acad. Sci. USA 87: 2466-2470, 1990[Abstract].

33.   Wayman, C. P., I. McFadzean, A. Gibson, and J. F. Tucker. Two distinct membrane currents activated by cyclopiazonic acid-induced calcium store depletion in single smooth muscle cells of the mouse anococcygeus. Br. J. Pharmacol. 117: 566-572, 1996[Abstract].

34.   Wayman, C. P., I. McFadzean, A. Gibson, and J. F. Tucker. Cellular mechanisms underlying carbachol-induced oscillations of calcium-dependent membrane current in smooth muscle cells from mouse anococcygeus. Br. J. Pharmacol. 121: 1301-1308, 1997[Abstract].

35.   Xuan, Y.-T., O.-L. Wang, and A. R. Whorton. Thapsigargin stimulates Ca2+ entry in vascular smooth muscle cells: nicardipine-sensitive and -insensitive pathways. Am. J. Physiol. Cell Physiol. 262: C1258-C1265, 1992[Abstract/Free Full Text].

36.   Yuan, X.-J. Role of calcium-activated chloride current in regulating pulmonary vasomotor tone. Am. J. Physiol. Lung Cell. Mol. Physiol. 272: L959-L968, 1997[Abstract/Free Full Text].

37.   Yuan, X.-J., T. Sugiyama, W. F. Goldman, L. J. Rubin, and M. P. Blaustein. A mitochondrial uncoupler increases KCa currents but decreases Kv currents in pulmonary artery myocytes. Am. J. Physiol. Cell Physiol. 270: C321-C331, 1996[Abstract/Free Full Text].

38.   Yule, D. I., E. T. Kim, and J. A. Williams. Tyrosine kinase inhibitors attenuate "capacitative" Ca2+ influx in rat pancreatic acinar cells. Biochem. Biophys. Res. Commun. 202: 1697-1704, 1994[ISI][Medline].


Am J Physiol Lung Cell Mol Physiol 278(1):L118-L130
0002-9513/00 $5.00 Copyright © 2000 the American Physiological Society