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(Circulation Research. 1997;81:812-823.)
© 1997 American Heart Association, Inc.

Articles

Phenylephrine-Induced Ca²⁺ Oscillations in Canine Pulmonary Artery Smooth Muscle Cells

Hiroshi Hamada, Derek S. Damron, Sung Jin Hong, David R. Van Wagoner, , Paul A. Murray

From the Center for Anesthesiology Research, Division of Anesthesiology and Critical Care Medicine, The Cleveland (Ohio) Clinic Foundation.

Correspondence to Paul A. Murray, PhD, Carl E. Wasmuth Chair and Director, Center for Anesthesiology Research-FF4, The Cleveland Clinic Foundation, 9500 Euclid Ave, Cleveland, OH 44195. E-mail murrayp{at}cesmtp.ccf.org

	Abstract

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Abstract Modulation of [Ca²⁺]_i in response to receptor activation is a critical determinant of vascular smooth muscle tone. In this study, we examined the effect of continuous stimulation of ₁-adrenoceptors with phenylephrine (PE) on [Ca²⁺]_i in single pulmonary artery smooth muscle cells (PASMCs) cultured from explants of canine intrapulmonary artery. Fura 2–loaded PASMCs pretreated with propranolol (5 µmol/L) were continuously superfused with PE at 37°C on the stage of an inverted fluorescence microscope, and [Ca²⁺]_i was measured using a dual-wavelength spectrofluorometer. Resting values of [Ca²⁺]_i were 96±4 nmol/L. PE (10 µmol/L) stimulated oscillations in [Ca²⁺]_i at a frequency of 1.35±0.07/min, which reached a peak [Ca²⁺]_i of 650±26 nmol/L (n=69 cells). The oscillations lasted for >30 minutes and were constant in amplitude and frequency. Both the amplitude and frequency of PE-induced [Ca²⁺]_i oscillations increased in a dose-dependent (3x10^-8 to 10^-4 mol/L) manner. Pretreatment with the ₁-adrenoceptor antagonist prazosin (50 nmol/L) or removal of extracellular Ca²⁺ abolished the repetitive [Ca²⁺]_i oscillations induced by PE. The voltage-operated Ca²⁺ channel blockers nifedipine (1 µmol/L) and verapamil (1 µmol/L) had no effect on the [Ca²⁺]_i oscillations. In contrast, inhibition of phospholipase C with U73122 (10^-7 to 10^-5 mol/L) attenuated the oscillations in a dose-dependent fashion. The nonselective protein kinase inhibitor staurosporine (10^-9 to 10^-7 mol/L) had a minimal inhibitory effect on the oscillations. Caffeine (30 mmol/L) and thapsigargin (1 µmol/L) abolished the oscillations, whereas pretreatment with ryanodine (1 to 100 µmol/L) had no effect. In freshly dispersed PASMCs, PE (10 µmol/L) induced oscillations in [Ca²⁺]_i similar to those observed in cultured cells, and patch-clamp experiments revealed oscillations in membrane potential. These results indicate that PE induces [Ca²⁺]_i oscillations in PASMCs via stimulation of ₁-adrenoceptors coupled to phospholipase C activation. Voltage-operated Ca²⁺ channels and protein kinases are not required for the oscillations. The requirement for extracellular Ca²⁺ and intracellular Ca²⁺ stores indicates that both Ca²⁺ influx and intracellular Ca²⁺ release play a role in the maintenance of the oscillations.

Key Words: Ca²⁺ • pulmonary artery • -adrenoceptor • phospholipase C

	Introduction

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An increase in [Ca²⁺]_i plays a primary role in triggering contraction of vascular smooth muscle cells. Increases in [Ca²⁺]_i result from an influx of Ca²⁺ across the sarcolemma and/or release of Ca²⁺ from intracellular stores. Although K⁺ channels play an important role in the maintenance of steady state vasomotor tone,^{1 2} agonists capable of increasing [Ca²⁺]_i in smooth muscle cells are important for the acute modulation of vasomotor tone in response to neural, humoral, and local activation.

Sympathetic ₁-adrenoceptors on smooth muscle cells are G protein–coupled receptors that mediate contraction by increasing [Ca²⁺]_i.³ These receptors are linked to the activation of phospholipase C, which hydrolyzes polyphosphatidylinositol 4,5-bisphosphate to IP₃ and DG.⁴ IP₃ can mobilize Ca²⁺ from intracellular stores to increase [Ca²⁺]_i, whereas DG can stimulate PKC,⁵ which may be involved in the maintenance of sustained contractions. The increase in [Ca²⁺]_i due to mobilization of Ca²⁺ from intracellular stores by IP₃ is transient because of the limited storage capacity of intracellular pools as well as the concomitant activation of membrane Ca²⁺ pumps, which restore [Ca²⁺]_i to resting levels. Repetitive oscillations in [Ca²⁺]_i in response to agonist stimulation have been demonstrated in several types of smooth muscle, which suggests that Ca²⁺ could exert its action through a frequency-dependent mechanism.^{6 7}

In the pulmonary circulation, ₁-adrenoceptor activation causes vasoconstriction in vivo.⁸ In isolated pulmonary arterial rings, increases in developed tension induced by ₁-adrenoceptor agonists are potentiated by removal of the endothelium.⁹ Therefore, acute changes in pulmonary vasomotor tone result from a balance between stimuli causing smooth muscle contraction and endothelium-derived vasoactive factors causing relaxation.¹⁰ Such interactions between endothelium and vascular smooth muscle make it difficult to assess the direct effects of vasoactive agents on Ca²⁺ signaling and smooth muscle cell contraction. Little is known about the direct effects of ₁-adrenoceptor stimulation on [Ca²⁺]_i in the pulmonary circulation at the cellular level. In the present study, we examined the direct effects of the ₁-adrenoceptor agonist phenylephrine on [Ca²⁺]_i at the single-cell level, in cultures of canine pulmonary arterial myocytes. PE stimulated persistent oscillations in [Ca²⁺]_i that were dependent on the presence of extracellular Ca²⁺ but did not require activation of VOCs. The amplitude and frequency of the oscillations increased in a dose-dependent manner with increasing concentrations of PE. The maintenance of the oscillations also depended on caffeine-sensitive intracellular stores of Ca²⁺ but did not involve Ca²⁺ release via the ryanodine release channel or the activation of protein kinases. We also observed PE-induced oscillations in [Ca²⁺]_i and membrane potential in freshly dispersed pulmonary artery smooth muscle cells. Oscillations in [Ca²⁺]_i in response to ₁-adrenoceptor activation may provide a frequency-dependent Ca²⁺ signal for acute modulation of pulmonary vasomotor tone.

	Materials and Methods

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Animals
Pulmonary arteries were isolated from adult male 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 fentanyl and 30 mg/kg IV pentobarbital sodium), with endotracheal intubation and positive-pressure ventilation. The dogs were killed by removing the mobilizable blood volume, followed by the administration of saturated KCl (30 mL). A thoracotomy was performed via the left fifth intercostal space. The heart and lungs were removed en bloc, and pulmonary arteries were isolated and dissected in a laminar flow hood under sterile conditions.

Cell Culture of PASMCs
Primary cultures of smooth muscle cells were obtained from segmental and subsegmental branches of the pulmonary artery (the third and fourth generation of branches from the main pulmonary artery having diameters of <4 mm). The intralobar arteries were carefully dissected and prepared for tissue culture. Explant cultures were prepared according to the method of Campbell and Campbell¹¹ with minor modifications. Briefly, the endothelium was removed by gently rubbing with a sterile cotton swab. The tunica adventitia was carefully removed, together with the most superficial part of the tunica media. The remaining part of the media was cut into 2-mm² pieces that were explanted in 25-cm² culture flasks. The explants were nourished by DMEM/F-12 medium containing 10% FBS and 1% antibiotic-antimycotic mixture solution (10 000 U/mL penicillin, 10 000 µg/mL streptomycin, and 25 µg/mL 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. Cells were allowed to grow for an additional 7 to 10 days until subconfluence was achieved. Cells were then subcultured nonenzymatically to 75-cm² culture flasks and/or 35-mm glass dishes specially designed for fluorescence microscopy (Bioptechs T system). Cells were used for experimentation within 72 hours.

Immunolabeling of PASMCs With Smooth Muscle -Actin
Confluent PASMCs were washed in PBS (three times for 5 minutes each) and then rapidly fixed in absolute acetone (-20°C) for 10 minutes. Fixed cells were rehydrated by washing in PBS (twice for 10 minutes) and then incubated with a fluorescein-labeled antibody against smooth muscle -actin (10 µg/mL, Sigma Chemical Co) for 2 hours at room temperature. Unbound antibody was removed by washing in PBS (twice for 10 minutes). Cells were costained with propidium iodide (20 µg/mL, Molecular Probes) for 1 hour at room temperature to localize the nucleus. Coverslips were mounted on glass slides using Vectashield (Vector Labs), and photomicrographs were taken on a Nikon Optiphot fluorescence microscope using Kodak PPF 400 color print film.

Freshly Dispersed PASMCs
PASMCs were isolated using a modification of the dispersion procedure described by Albarwani et al.¹² The isolated lungs were stored at room temperature in solution A (Table). Pulmonary arteries were rapidly dissected and stored briefly (30 minutes) at 4°C in this solution. A segment (5 cm) of pulmonary artery was opened with surgical scissors, and the endothelium was removed by gently rubbing the surface with a cotton swab. The tissue was then transferred to a 15-mL centrifuge tube containing 10 mL of the initial digestion solution (solution B) supplemented with 15 mg papain (14 U/mg) and 10 mg dithiothreitol. The tissue was placed in this solution for 15 minutes at 4°C and then transferred to a shaking water bath (37°C) for 6 minutes. The tissue was then removed and minced into 1-cm segments. The minced tissue was placed in a 125-mL Erlenmeyer flask containing 10 mL of solution B supplemented with 0.75 mg/mL collagenase (Worthington type I, 307 U/mg), 0.5 mg/mL trypsin inhibitor (Sigma type II), and 10 mmol/L butanedione monoxime. The minced tissue was shaken in this solution in the water bath for 6 minutes. The tissue and solution were then triturated three or four times with a plastic transfer pipette and transferred to a plastic beaker. Fresh collagenase solution (10 mL) was added to the Erlenmeyer flask. The tissue was transferred back into the fresh collagenase solution, and the first digestion solution (containing the released PASMCs) was gently centrifuged (10g) for 1 minute. The supernatant (8 mL) was aspirated and discarded, and the remaining 2 mL was washed and resuspended in solution C, supplemented with 1% BSA (Sigma fraction V). The tissue digestion procedure was repeated three or four times. The PASMCs were maintained in solution C at room temperature and gassed with 100% O₂ until they were studied (within 2 to 5 hours of isolation). Yields of spindle-shaped PASMCs were in the range of 10% to 30%. All chemicals and enzymes were obtained from Sigma, except the collagenase. All plastic and glassware were pretreated with Sigmacote.

View this table: [in this window] [in a new window]   Table 1. Solutions

Patch-Clamp Recordings in PASMCs
Conventional whole-cell recording techniques were used to measure voltage-gated Ca²⁺ currents in freshly dispersed PASMCs. Aliquots of the cell suspension were transferred to a 35-mm dish on the stage of an inverted microscope, maintained at a temperature of 35°C (Bioptech's T system), and gassed with 100% O₂. Cells were superfused (1 to 2 mL/min) with a control cell bath (solution D). Sylgard-coated low-resistance (2- to 3-M) electrodes were filled with a CsCl pipette solution (solution E). Data acquisition was performed with pClamp 6.0 software controlling an Axopatch 200A amplifier (Axon Instruments). Whole-cell currents were recorded from a holding potential of -50 mV (to inactivate voltage-dependent Na⁺ current). Once stable control current recordings were obtained, the superfusion solution was switched to one containing 10 µmol/L nifedipine. Leak current was corrected by digitally subtracting the current after nifedipine exposure from the control current recording. Current densities (pA/pF) were obtained by dividing current amplitudes by the capacitance value. Series resistance values were typically in the range of 5 to 6 M and were not corrected.

Current-clamp experiments were performed using the nystatin–perforated patch technique. The experimental setup was nearly identical to that used to record Ca²⁺ currents; however, recordings were obtained using an Axopatch 1C amplifier in the current-clamp mode. Sylgard-coated low-resistance electrodes were front-filled with a pipette solution (solution F) by capillary action and then back-filled with a similar solution to which nystatin (final concentration, 100 mg/mL) had been added from a stock solution made fresh daily. Once nystatin was added to the buffer, the pipette solution was sonicated (30 seconds) and used within 3 hours. After a stable seal was formed in the voltage-clamp mode, the amplifier was switched to current-clamp mode. Membrane potential was monitored and recorded using Axotape software (Axon Instruments).

Fura 2–Loading Procedure
Twenty-four hours before experimentation, the culture medium containing 10% FBS was substituted for the serum-free medium to arrest cell growth, allow for establishment of steady state cellular events independent of cell division, and prevent a false estimate of [Ca²⁺]_i due to binding of available dye by a high serum protein concentration in the medium.¹³ PASMCs were washed twice in LB, which contained (mmol/L) NaCl 125, KCl 5, MgSO₄ 1.2, glucose 11, CaCl₂ 1.8, and HEPES 25, plus 0.2% BSA, at pH 7.40 adjusted with NaOH. PASMCs were then incubated in LB containing 2 µmol/L fura 2-AM, the acetoxymethyl derivative of fura 2 (Molecular Probes), at ambient temperature for 30 minutes. After the 30-minute loading period, the cells were washed twice in LB and incubated at ambient temperature for an additional 20 minutes before the study. This provided sufficient time to wash away any extracellular fura 2-AM and for intracellular esterases to cleave fura 2-AM into the active fura 2.¹⁴

Freshly dispersed PASMCs were loaded with fura 2-AM in a similar fashion. Freshly dispersed PASMCs were washed twice with LB and then incubated with LB containing fura 2-AM (2 µmol/L) at room temperature for 30 minutes. Cells were washed twice with LB (twice for 10 minutes) to remove extracellular fura 2-AM.

Determination of [Ca²⁺]_i
Culture dishes containing fura 2–loaded PASMCs were placed in a temperature-regulated (37°C) chamber (Bioptechs, Inc) mounted on the stage of an Olympus IX-70 inverted fluorescence microscope. Fluorescence measurements were performed on individual smooth muscle cells in a culture monolayer or in freshly dispersed cells using a dual-wavelength spectrofluorometer (Deltascan RFK6002, Photon Technology Intl) 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 (mmol/L) NaCl 125, KCl 5, MgSO₄ 1.2, glucose 11, CaCl₂ 2.5, and HEPES 25, at 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 was obtained and subtracted automatically from the subsequent experimental measurement. Fura 2 fluorescence signals (340, 380, and 340/380 ratio) originating from individual PASMCs were continuously monitored at a sampling frequency of 25 Hz and were collected and stored using a software package from Photon Technology Intl (Felix).

Fura 2 Titration
Estimations of [Ca²⁺]_i were achieved by comparing the cellular fluorescence ratio with fluorescence ratios acquired using fura 2 (free acid) in buffers containing known [Ca²⁺]. [Ca²⁺]_i was calculated as described by Grynkiewicz et al.¹⁵

Data Analysis
The amplitude and frequency of PE-induced increases in [Ca²⁺]_i were measured in individual PASMCs. Amplitude was calculated by averaging the peak ratio value obtained for each oscillation before and after the intervention (four or five oscillations). The change in the 340/380 fluorescence ratio was then calculated by subtracting the resting ratio value (baseline). Changes in the amplitude of the 340/380 fluorescence ratio after interventions were also expressed as percentage of control. PASMCs began to oscillate [Ca²⁺]_i in response to PE at a concentration of 3x10^-8 mol/L, so the amplitude and frequency of the oscillations at this concentration were considered the control response and were defined as 100% of control. 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. Results are expressed as mean±SEM. Statistical analysis was performed using repeated-measures ANOVA followed by Bonferroni/Dunn post hoc testing. Differences were considered statistically significant at P<.05.

	Results

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Immunolabeling With Smooth Muscle -Actin
Indirect immunofluorescent staining of the cultured PASMCs demonstrates the characteristic lace and longitudinal fluorescent patterns of smooth muscle cell actin filaments (Fig 1). Propidium iodide was used to identify the nucleus. For the first through third subcultured passages, canine PASMCs were >99% pure, as determined with indirect immunofluorescent staining.

View larger version (137K): [in this window] [in a new window]   Figure 1. Cultured PASMCs show positive staining for smooth muscle -actin. Propidium iodide was used to label the nucleus. Magnification x400.

Effect of ₁-Adrenoceptor Stimulation With PE on [Ca²⁺]_i in PASMCs
Resting values of [Ca²⁺]_i were 96±4 nmol/L. PASMCs were pretreated with the ß-adrenoceptor antagonist propranolol (5 µmol/L) to eliminate any ß-agonist effect of PE. Continuous superfusion of PE (10^-5 mol/L), known to be a maximally effective concentration,^{16 17} stimulated repetitive [Ca²⁺]_i oscillations at a frequency of 1.35±0.07 transients/min for >30 minutes (Fig 2, top). The [Ca²⁺]_i reached an average peak value of 650±26 nmol/L. Both amplitude and frequency of the [Ca²⁺]_i oscillations in response to PE were dose dependent (Fig 2, bottom; n=10 cells). The amplitude of the individual [Ca²⁺]_i oscillations reached a plateau at a concentration of 10^-5 mol/L PE (265±26% of control), whereas the frequency was still increasing even at 10^-4 mol/L PE (348±14%). The frequency and amplitude of PE-induced [Ca²⁺]_i oscillations were similar in cells through the third passage.

View larger version (33K): [in this window] [in a new window]   Figure 2. Effect of PE on [Ca2+]i in individual PASMCs. Top, A representative trace demonstrating that PE (10 µmol/L) stimulated persistent oscillations in [Ca2+]i lasting >30 minutes. PE was added to the superfusion buffer at the arrow. Bottom, Summarized data depicting the dose-dependent increases in both amplitude and frequency of [Ca2+]i oscillations induced by PE (n=10 cells).

Effect of ₁-Adrenoceptor Inhibition With Prazosin
To determine whether the [Ca²⁺]_i oscillations were mediated by ₁-adrenoceptor activation, the ₁-adrenoceptor antagonist prazosin (50 nmol/L) was added to the superfusate after induction of [Ca²⁺]_i oscillations in response to PE (10 µmol/L). Prazosin completely abolished PE-induced [Ca²⁺]_i oscillations (Fig 3, n=14 cells).

View larger version (17K): [in this window] [in a new window]   Figure 3. A representative trace demonstrating the effect of the 1-adrenoceptor antagonist prazosin on [Ca2+]i oscillations induced by PE (10 µmol/L). Prazosin (50 nmol/L) was added to the superfusion buffer as indicated.

Effect of Removal of Extracellular Ca²⁺
We examined the extent to which extracellular Ca²⁺ contributed to PE-induced [Ca²⁺]_i oscillations in PASMCs. After initiation of PE-induced (10 µmol/L) [Ca²⁺]_i oscillations, PE-containing buffer was washed out and replaced with Ca²⁺-free Krebs-Ringer buffer containing EGTA (100 µmol/L). After 3 minutes of incubation in the absence of extracellular Ca²⁺, PE was added back to the superfusion buffer. Although in some cells PE stimulated several transient oscillations in [Ca²⁺]_i (Fig 4, top), sustained oscillations were not observed in the absence of extracellular Ca²⁺ (n=16 cells). Readdition of Ca²⁺ to the superfusion buffer in the presence of PE resulted in an immediate recovery of the repetitive [Ca²⁺]_i oscillations (Fig 4, top).

View larger version (38K): [in this window] [in a new window]   Figure 4. Requirement of extracellular Ca2+ for the [Ca2+]i oscillations induced by PE. Top, A representative trace depicting the effect of PE (10 µmol/L) on [Ca2+]i oscillations following removal of extracellular Ca2+. Repetitive [Ca2+]i oscillations were abolished when the external Ca2+ was removed (Ca2+-free buffer plus 100 µmol/L EGTA). [Ca2+]i oscillations returned when Ca2+ was added back to the superfusate. Bottom, Summarized data depicting the effect of voltage-operated Ca2+ channel blockers nifedipine (NIF, 1 µmol/L) or verapamil (VER, 1 µmol/L) on [Ca2+]i oscillations induced by PE (10 µmol/L) compared with control (CTRL). [Ca2+]i oscillations persisted in the presence of NIF or VER (n=16 cells).

Effect of Voltage-Operated Ca²⁺ Channel Blockers
To examine whether the requirement of extracellular Ca²⁺ for the PE-induced [Ca²⁺]_i oscillations involved activation of VOCs, the L-type VOC antagonists, verapamil or nifedipine, were added to the superfusate in the presence of PE. Neither verapamil (1 µmol/L) nor nifedipine (1 µmol/L) altered the amplitude or frequency of the PE-induced (10 µmol/L) [Ca²⁺]_i oscillations (Fig 4, bottom; n=16 cells).

Effect of ₁-Adrenoceptor Stimulation With PE on [Ca²⁺]_i and Membrane Potential in Freshly Dispersed PASMCs
In order to determine whether the Ca²⁺ oscillations induced by PE in the cultured cells were also present in "native" cells, we developed a method for isolating freshly dispersed, Ca²⁺-tolerant, spindle-shaped PASMCs. These cells are more suitable for patch-clamp analysis and allowed us to examine electrophysiological changes in response to ₁-adrenoceptor activation with PE. A representative myocyte isolated with this protocol is shown in Fig 5A. Resting values of [Ca²⁺]_i were 75±8 nmol/L in freshly dispersed PASMCs (n=9 cells). Exposure to PE (10 µmol/L) resulted in [Ca²⁺]_i oscillations at a frequency of 0.89±0.09 transients/min, and the average peak value of [Ca²⁺]_i reached 293±13 nmol/L (Fig 5B). Nifedipine (10 µmol/L) had no effect on PE-induced [Ca²⁺]_i oscillations in these freshly dispersed PASMCs.

View larger version (50K): [in this window] [in a new window]   Figure 5. In freshly dispersed PASMCs, PE initiates oscillations in [Ca2+]i and membrane potential. A, A typical myocyte isolated from canine pulmonary artery using the dispersion protocol outlined in "Materials and Methods" is shown. B, [Ca2+]i oscillations in freshly dispersed myocytes are qualitatively similar to those observed in cultured PASMCs. C, Application of PE (10 µmol/L) to a PASMC monitored under current-clamp conditions causes a depolarization and initiation of rapid oscillations in membrane potential (Em).

We also measured the effects of PE on membrane potential in freshly dispersed PASMCs. The mean resting potential was -45±3 mV (n=16) before the addition of PE. Addition of PE (10 µmol/L) caused a rapid depolarization followed by oscillations in membrane potential (Fig 5C). Nifedipine (10 µmol/L) had no effect on the PE-induced oscillations in membrane potential. The effects of PE on [Ca²⁺]_i oscillations (Fig 5B) and membrane potential (Fig 5C) were measured in separate experiments from cells isolated from the same lung.

We also performed experiments in the freshly dispersed PASMCs to determine whether dihydropyridine-sensitive Ca²⁺ currents were present. Fig 6 illustrates typical Ca²⁺ currents recorded from an isolated, freshly dispersed PASMC before and after superfusion with 10 µmol/L nifedipine. Similar recordings were obtained in six additional freshly dispersed PASMCs. Peak current density was measured in each myocyte and normalized to myocyte capacitance. The mean peak Ca²⁺ current density was -2.06±0.54 pA/pF (n=7), with a mean capacitance of 15±2 pF.

View larger version (29K): [in this window] [in a new window]   Figure 6. L-type Ca2+ currents are present in freshly dispersed PASMCs. A, Raw current traces recorded from a holding potential of –50 mV using 75-millisecond voltage steps to potentials ranging between –40 and +30 mV, in 10-mV increments, applied at 3-second intervals. B, Currents elicited in the same PASMC using the same voltage-clamp protocol after 2-minute superfusion with 10 µmol/L nifedipine. C, Nifedipine-sensitive current obtained by subtracting the current recorded after nifedipine treatment from the current obtained before nifedipine was added to the bath. D, Current-voltage relationship for the inward Ca2+ current before () and after () application of nifedipine to the bath.

Effect of Phospholipase C Inhibition
The activation of ₁-adrenoceptors generates two intracellular messengers, IP₃ and DG, via a G protein–dependent phospholipase C transduction pathway.⁴ We used U73122, an inhibitor of the phospholipase C family,¹⁸ to investigate the link between PE-induced (10 µmol/L) [Ca²⁺]_i oscillations and the formation of IP₃ and DG in PASMCs. U73122 caused dose-dependent inhibition of both amplitude and frequency of PE-induced [Ca²⁺]_i oscillations (Fig 7, top and bottom). Thapsigargin increases [Ca²⁺]_i by decreasing its uptake into the SR via inhibition of the Ca²⁺-ATPase.¹⁹ Addition of thapsigargin following pretreatment with U73122 caused a rapid increase in [Ca²⁺]_i, indicating that the inhibitory effect of U73122 was not due to interference with the storage or release of Ca²⁺ from intracellular stores (Fig 7, top; n=13 cells).

View larger version (32K): [in this window] [in a new window]   Figure 7. Effect of phospholipase C inhibition with U73122 on [Ca2+]i oscillations induced by PE. Top, A representative trace depicting the dose-dependent inhibitory effect of U73122 on [Ca2+]i oscillations induced by PE (10 µmol/L). Increasing concentrations of U73122 (0.1 to 10 µmol/L) were added to the superfusion buffer (indicated by the down arrows) in the continued presence of PE. The SR Ca2+-ATPase inhibitor thapsigargin (Tg) was added to demonstrate the presence of intracellular Ca2+ stores during U73122 treatment. Bottom, Summarized data showing the dose-dependent inhibitory effect of U73122 on both amplitude and frequency of the [Ca2+]i oscillations induced by PE (n=13 cells).

Effect of Protein Kinase Inhibition
The inhibitory effect of U73122 suggests that IP₃ and/or DG is likely to play a key role in PE-induced Ca²⁺ oscillations in PASMCs. IP₃ mobilizes Ca²⁺ from the SR, whereas DG activates PKC. We used staurosporine, a potent nonselective inhibitor of protein kinases, to examine the extent to which protein kinase activation is involved in regulating [Ca²⁺]_i oscillations induced by PE. Fig 8 demonstrates that staurosporine inhibits both the amplitude and frequency of PE-induced [Ca²⁺]_i oscillations in the range of 3 to 100 nmol/L (IC₅₀ for PKC, 8 nmol/L).²⁰ However, the inhibitory effect of staurosporine was small, even at the highest concentration used in the present study (amplitude, 88±2%; frequency, 92±2% of control values; n=12 cells).

View larger version (19K): [in this window] [in a new window]   Figure 8. Summarized data showing the effect of the nonselective protein kinase inhibitor staurosporine on [Ca2+]i oscillations induced by PE. Increasing concentrations of staurosporine were added to the superfusate in the continued presence of 10 µmol/L PE (n=12 cells).

Role of SR Ca²⁺
IP₃, another intracellular second messenger in the phospholipase C signal transduction pathway, increases [Ca²⁺]_i by stimulating Ca²⁺ release from the SR via the IP₃ receptor. In addition, Ca²⁺ influx from extracellular sources can trigger SR Ca²⁺ release via the ryanodine receptor by a process known as Ca²⁺-induced Ca²⁺ release. We examined the role of the SR in PE-induced [Ca²⁺]_i oscillations. Caffeine and ryanodine were used as tools to study SR Ca²⁺ release pathways and to investigate the role of the SR in PE-induced [Ca²⁺]_i oscillations. Caffeine (30 mmol/L) alone had no effect on resting [Ca²⁺]_i. However, [Ca²⁺]_i oscillations in response to PE were completely inhibited when caffeine was added to the superfusate, and the oscillations returned when caffeine was washed out (Fig 9). The membrane-permeabilizing agent, ionomycin (5 µmol/L), was used to investigate the efficacy of caffeine to deplete SR Ca²⁺ stores. In the absence of extracellular Ca²⁺, any increase in the fluorescence signal in response to ionomycin should reflect the release of Ca²⁺ from intracellular stores. Fig 10, top, demonstrates that in the absence of extracellular Ca²⁺, ionomycin stimulates a large [Ca²⁺]_i transient, reaching a peak fluorescence ratio of 10.3±0.9 (n=11 cells). Fig 10, bottom, shows that although caffeine abolishes PE-induced [Ca²⁺]_i oscillations, ionomycin stimulates a [Ca²⁺]_i transient similar in magnitude to that observed in the absence of caffeine (peak fluorescence ratio, 10.5±0.7; n=8 cells).

View larger version (23K): [in this window] [in a new window]   Figure 9. A representative trace depicting the effect of caffeine on [Ca2+]i oscillations induced by PE. Caffeine (30 mmol/L) was added to the superfusion buffer in the continued presence of PE (10 µmol/L). After washout of caffeine, PE-induced oscillations in [Ca2+]i returned. No change in baseline [Ca2+]i was observed in response to caffeine itself (n=12 cells).

View larger version (20K): [in this window] [in a new window]   Figure 10. Effect of caffeine (Caff) on ionomycin (Iono)–releasable Ca2+ stores in PASMCs. Top, A typical trace depicting the effect of Iono on [Ca2+]i in a single PASMC. PE was washed out of the bath using Ca2+-free buffer containing EGTA (1 mmol/l). Iono (5 µmol/L) was added where indicated on the graph. Bottom, A typical trace depicting the effect of Iono on [Ca2+]i in a single PASMC after pretreatment with Caff. After addition of Caff (30 mmol/L), the cell was perfused with Ca2+-free buffer containing both EGTA (1 mmol/L) and Caff (10 mmol/L). Iono (5 µmol/L) was added where indicated in the figure.

Ryanodine was also used to investigate the role of the SR Ca²⁺ store. After establishment of PE-induced [Ca²⁺]_i oscillations, PE was washed out, and ryanodine (1 to 100 µmol/L) was added. In some cells (8 of 13 studied), addition of ryanodine (5 µmol/L) resulted in a slow increase in the resting level of [Ca²⁺]_i in PASMCs (Fig 11, top), whereas in other cells (5 of 13 studied), no increase was observed. In either case, when PE was reintroduced into the bath in the continued presence of ryanodine, [Ca²⁺]_i oscillations returned and were not significantly different in amplitude or frequency compared with the [Ca²⁺]_i oscillations observed in the absence of ryanodine (Fig 11, bottom). Higher concentrations of ryanodine (100 µmol/L) did not alter baseline [Ca²⁺]_i and had no effect on the oscillations. Ryanodine (5 µmol/L), like caffeine (Fig 10, bottom) had no significant inhibitory effect on ionomycin-releasable Ca²⁺ stores (Fig 12).

View larger version (30K): [in this window] [in a new window]   Figure 11. Effect of ryanodine (Ryan) on [Ca2+]i oscillations induced by PE. Top, A typical trace depicting the effect of ryanodine (5 µmol/L) alone on baseline [Ca2+]i and in the presence of PE-induced (10 µmol/L) [Ca2+]i oscillations. In the presence of PE-induced [Ca2+]i oscillations, Ryan (5 and 100 µmol/L) was added to the superfusate (n=13 cells). Bottom, Summarized data showing the effect of Ryan on amplitude and frequency of [Ca2+]i oscillations induced by PE.

View larger version (12K): [in this window] [in a new window]   Figure 12. Effect of ryanodine on ionomycin (Iono)–releasable Ca2+ stores in PASMCs. In the presence of extracellular Ca2+ (2.5 mmol/L), cells were treated with vehicle (dimethyl sulfoxide [DMSO]) (top) or ryanodine (5 µmol/L) (bottom) for 5 minutes where indicated in the figure. The buffer was then rapidly exchanged to a Ca2+-free buffer, followed by addition of Iono (5 µmol/L) as indicated. Similar results were obtained in six cells.

We also examined whether inhibition of the SR Ca²⁺ pump with thapsigargin altered PE-induced [Ca²⁺]_i oscillations. Fig 13 demonstrates that addition of thapsigargin (1 µmol/L) in the continued presence of PE resulted in an immediate increase in the fluorescence ratio, which only partially returned to baseline. In addition, the [Ca²⁺]_i oscillations induced by PE were abolished. Similar results were observed in nine cells.

View larger version (19K): [in this window] [in a new window]   Figure 13. Effect of thapsigargin (Tg) on [Ca2+]i oscillations induced by PE. A typical trace depicting the effect of Tg (1 µmol/L) on PE-induced (10 µmol/L) [Ca2+]i oscillations. In the presence of PE-induced [Ca2+]i oscillations, Tg (1 µmol/L) was added to the superfusate. Similar results were obtained in nine cells.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The present study demonstrates that PE stimulates constant, repetitive, transient increases in [Ca²⁺]_i (Ca²⁺ oscillations) in cultured and freshly dispersed canine PASMCs. These oscillations are mediated through ₁-adrenoceptor activation, as demonstrated by the inhibition of the oscillations by the ₁-adrenoceptor antagonist prazosin. The Ca²⁺ oscillations require extracellular Ca²⁺ but do not require the activation of VOCs. Both the amplitude and the frequency of the PE-induced Ca²⁺ oscillations are dose dependent. Phospholipase C inhibition reduces the Ca²⁺ oscillations in a dose-dependent manner. The maintenance of the oscillations also depends on IP₃-dependent stores of Ca²⁺ and the activity of the SR Ca²⁺ pump but does not involve Ca²⁺ release via the ryanodine release channel or activation of protein kinases.

Use of Cultured Cells to Study Ca²⁺ Signaling in Vascular Smooth Muscle
It is well known that vascular smooth muscle cells undergo spontaneous changes in their morphological and functional properties when grown in culture.²¹ Cultured cells may be classified into either a nonproliferating "contractile" state or a proliferating "synthetic" state.²² In addition to these structural changes, alterations in Ca²⁺ signaling are also known to occur in cultured cells.²³ Therefore, studies using cultured vascular smooth muscle cell models to investigate Ca²⁺ signaling may not necessarily represent the Ca²⁺ signaling pathways that are active in native cells. However, because both phenotypes are also found in the vessel wall after vascular injury and in atherosclerotic lesions,²⁴ studies carried out in both cultured and/or freshly dispersed cells can provide important information about cellular regulation in vascular smooth muscle. Cultured PASMCs were predominantly used in the present study. In addition, we used freshly dispersed PASMCs to confirm the physiological relevance of our findings.

PE-Induced Ca²⁺ Oscillations Are Dose Dependent
The Ca²⁺ response to ₁-adrenoceptor activation reported in the present study differs from that observed in rat tail arterial smooth muscle cells and cat pulmonary arterial myocytes, where ₁-adrenoceptor activation induced an initial peak, followed by a sustained plateau of [Ca²⁺]_i.^{25 26} A recent study using freshly dispersed rat PASMCs demonstrated that angiotensin II stimulated three to six Ca²⁺ oscillations of constant duration but of decreasing amplitude,⁷ a pattern different from that observed in the present study. The pattern of Ca²⁺ oscillations observed in the present study is similar, although not identical, to the oscillations reported in various other cell types, such as rat hepatocytes,²⁷ endothelial cells,²⁸ and human uterine arterial smooth muscle cells.²⁹ The Ca²⁺ oscillations in those cell types were comparable in frequency (0.7 to 1.4 min^-1) and in peak [Ca²⁺]_i (500 to 700 nmol/L) to those observed in the present study. The frequency of oscillations has been shown to depend on external conditions, particularly the concentration of the agonist,^{27 29 30} although it has been reported that angiotensin II–induced Ca²⁺ oscillations were independent of the agonist concentration.⁷ Our results clearly demonstrate that the amplitude and frequency of PE-induced Ca²⁺ oscillations increased in a dose-dependent manner in PASMCs.

Extracellular Ca²⁺ Is Required
PE-induced Ca²⁺ oscillations in canine PASMCs depend on extracellular Ca²⁺, because removal of external Ca²⁺ by EGTA resulted in an inhibition of repetitive Ca²⁺ oscillations. Extracellular Ca²⁺ was also required for the maintenance of Ca²⁺ oscillations induced by ATP in porcine aortic smooth muscle cells³¹ and by histamine in human endothelial cells.²⁸ In contrast, angiotensin and histamine induced Ca²⁺ oscillations in the absence of extracellular Ca²⁺ in systemic arterial smooth muscle cells.^{7 29} Similar results were observed in BC3H-1 cells in response to PE and histamine.⁶ Therefore, depending on the cell type and the agonist, some cells require extracellular Ca²⁺ for the oscillatory behavior, and some do not. Because Ca²⁺ oscillations in the present study slowly diminished after removal of extracellular Ca²⁺ from the superfusion buffer, it is possible that extracellular Ca²⁺ serves as a source of Ca²⁺ for priming or refilling of the intracellular Ca²⁺ stores.

VOCs Are Not Involved
Despite our electrophysiological confirmation that VOCs are present in PASMCs, neither verapamil nor nifedipine altered the PE-induced Ca²⁺ oscillations. These results indicate that VOCs are not involved in the regulation of these oscillations and that extracellular Ca²⁺ must enter the cytosol via another route, such as receptor-operated Ca²⁺ channels or a passive leak. Agonist-induced Ca²⁺ influx was not blocked by dihydropyridines in DDT₁ smooth muscle cells³² or in porcine aortic smooth muscle cells.³¹ Neither verapamil nor nicardipine had any significant effect on spontaneous Ca²⁺ oscillations in guinea pig ileum smooth muscle cells.³³ In contrast, spontaneous Ca²⁺ oscillations in A7r5 cells were abolished by the addition of nimodipine, which suggests that Ca²⁺ entry through VOCs was required for Ca²⁺ oscillations in that cell type.³⁴

Ca²⁺ Oscillations Depend on Phospholipase C Activation
Inhibition of the oscillations by U73122 indicates that IP₃ and/or DG, the product of phospholipase C activation, is involved in mediating these oscillations. This finding is similar to a previous study in which U73122 inhibited Ca²⁺ oscillations elicited by cholecystokinin and carbachol in pancreatic acinar cells.¹⁸ Thapsigargin has been shown to increase [Ca²⁺]_i by inhibiting the Ca²⁺-ATPase on intracellular stores¹⁹ and has been used to demonstrate specificity of U73122 actions in pancreatic acinar cells.¹⁸ Inhibition of the SR Ca²⁺ pump with thapsigargin resulted in an increase in [Ca²⁺]_i in the presence of U73122, which indicates that U73122 did not interfere with the storage or release of intracellular Ca²⁺. These results indicate that the initiation and/or maintenance of Ca²⁺ oscillations induced by PE could be mediated either by IP₃ and/or activation of PKC by DG.

Protein Kinases Are Not Involved
Activation of PKC can result in phosphorylation of specific substrate proteins, including ion channels and receptors,³⁵ as well as contractile proteins.³⁶ PE has been shown to elicit translocation of PKC and shortening in ferret vascular smooth muscle cells.³⁷ Prolongation of the falling phase of each Ca²⁺ spike induced by PE or vasopressin has been reported in staurosporine-pretreated rat hepatocytes, suggesting the involvement of PKC in the negative-feedback control of Ca²⁺ spiking.³⁸ In contrast, Ca²⁺ oscillations were observed in fibroblasts (REF52 cell line) in which PKC was downregulated with phorbol esters, indicating that PKC was not essential for the maintenance of these oscillations.³⁹ In the present study, the nonspecific kinase inhibitor, staurosporine,⁴⁰ inhibited both the amplitude and frequency of Ca²⁺ oscillations. However, this inhibitory effect was small (<10%), even at the highest concentration of staurosporine used in the present study. Therefore, it is unlikely that activation of any kinase, including PKC, plays a prominent role as a regulator of Ca²⁺ oscillations induced by PE in PASMCs.

Ca²⁺ Oscillations Depend on Caffeine-Sensitive Intracellular Ca²⁺ Release
Our results suggest that although extracellular Ca²⁺ is a prerequisite for the maintenance of the oscillations, IP₃-stimulated Ca²⁺ release from the SR may be the key messenger that generates Ca²⁺ oscillations in canine PASMCs. Our observation that caffeine reversibly abolished PE-induced Ca²⁺ oscillations suggests the involvement of SR Ca²⁺ stores. These findings are consistent with caffeine-inhibiting Ca²⁺ spike generation evoked by IP₃ in mouse pancreatic acinar cells⁴¹ and in Xenopus oocytes.⁴² However, caffeine itself did not increase [Ca²⁺]_i in PASMCs. These findings are in contrast to caffeine-triggered Ca²⁺ responses in smooth muscle cells isolated from rat pulmonary artery,⁷ as well as bovine and porcine coronary artery,⁴³ yet are similar to responses measured in other types of cultured vascular smooth muscle cells.^{44 45} This raises the question as to whether SR Ca²⁺ stores were effectively depleted by caffeine or whether caffeine has some other mechanism of action. The presence of ionomycin-releasable Ca²⁺ stores after caffeine pretreatment suggests that caffeine does not deplete intracellular Ca²⁺ stores in PASMCs. Caffeine has been shown to dose-dependently inhibit IP₃-gated channel activity in cerebellar and skeletal muscle microsomes, with complete inhibition occurring with 10 mmol/L caffeine (Bezprozvanny et al⁴⁶ ). A decrease in IP₃ binding was also observed in the study of Bezprozvanny et al. In pancreatic acinar cells, caffeine inhibited agonist-induced production of IP₃, with maximal inhibition occurring with 10 mmol/L caffeine.⁴⁷ Similar results have been obtained in PC12 cells.⁴⁸ These results suggest that caffeine may have an inhibitory effect on phospholipase C activity or on some intermediate step in the coupling between receptor stimulation and phospholipase C activation. Therefore, the effects of caffeine may be less specific than previously expected, and caffeine may have effects on IP₃ release and/or IP₃ binding in cultured PASMCs.⁴⁹

Ryanodine Does Not Inhibit Ca²⁺ Oscillations
Ryanodine was used to gain further insight into the role of SR Ca²⁺ stores in PE-induced Ca²⁺ oscillations. Ryanodine, which opens or closes its receptor/channel on SR depending on the concentration,⁵⁰ stimulated a slow increase in [Ca²⁺]_i at a concentration of <10 µmol/L in 60% of the cells studied, suggesting that the ryanodine-sensitive Ca²⁺ store exists in canine PASMCs. The net effect of ryanodine on [Ca²⁺]_i depends on other intracellular Ca²⁺–removal processes, including the sarcolemmal Ca²⁺ pump and the Na⁺-Ca²⁺ exchanger. It is possible that these intracellular Ca²⁺–removing processes were unable to keep up with the Ca²⁺ being released from the SR in response to ryanodine treatment in PASMCs, which could account for the sustained increase in baseline [Ca²⁺]_i. A ryanodine-induced sustained increase in [Ca²⁺]_i has been reported in both cardiac⁵¹ and smooth muscle^{43 52} preparations. In coronary artery smooth muscle cells,⁴³ ryanodine (10 µmol/L) caused a sustained increase in [Ca²⁺]_i above baseline. This effect was found to be due to both ryanodine-induced release of Ca²⁺ from the SR and inhibition of Ca²⁺ efflux via a reduction in the activity of the Na⁺-Ca²⁺ exchanger.⁴³ Thus, the sustained increase in [Ca²⁺]_i observed in most cells in the present study is consistent with previously reported effects of ryanodine in vascular smooth muscle cells.

Despite variability in the actions of ryanodine on baseline Ca²⁺, ryanodine (1 to 100 µmol/L) had no effect on either the frequency or amplitude of PE-induced Ca²⁺ oscillations when PE was reintroduced into the perfusate. These data suggest that either ryanodine-sensitive stores are not involved in the oscillations or that in the presence of extracellular Ca²⁺, the SR may be capable of refilling sufficiently rapidly to keep up with ryanodine-induced SR Ca²⁺ loss through channels locked in their subconductance state. Alternatively, ryanodine release channels may be scarce in cultured PASMCs. In the present study, ryanodine (5 µmol/L) failed to alter Ca²⁺ oscillations and ionomycin-releasable Ca²⁺ pools when extracellular Ca²⁺ was present. Because the oscillations were dependent on the presence of extracellular Ca²⁺ (Fig 4), a protocol to test the actions of ryanodine on Ca²⁺ oscillations or ionomycin-releasable Ca²⁺ stores in the absence of extracellular Ca²⁺ was not feasible. These data do not rule out the possibility of rapid refilling of the SR from extracellular Ca²⁺ sources but more likely support our caffeine data and the notion that there are not many ryanodine-sensitive channels present in cultured PASMCs. This would be consistent with the findings of others demonstrating the loss of caffeine-triggered Ca²⁺ transients in smooth muscle cells after only 3 days in culture.²³ Under culture conditions, the relative importance of Ca²⁺-induced Ca²⁺ release may be reduced, and it is unlikely that many ryanodine-sensitive Ca²⁺ release channels are present in these PASMCs.

Confirmation that the SR is directly involved in the Ca²⁺ oscillations is provided by the results obtained with thapsigargin. Thapsigargin, which blocks the SR Ca²⁺-ATPase, resulting in the inability of the SR to sequester Ca²⁺, completely blocked the Ca²⁺ oscillations. These results suggest that refilling of a SR Ca²⁺ pool by the Ca²⁺-ATPase is required for maintenance of the oscillations. Therefore, it appears that the ryanodine receptor, if present, plays a minor role in the oscillations, whereas caffeine-sensitive Ca²⁺ stores and refilling of these stores by the SR Ca²⁺-ATPase may play a major role in the oscillations.

Ca²⁺ Oscillations in Cell Signaling
Although speculative, it is possible that individual cell types display unique oscillatory patterns ("fingerprints" or "signatures"), which may represent an encoded signal to alter cell function.³⁰ The frequency of Ca²⁺ oscillations could be an important factor in the modulation of smooth muscle cell contractility and vasomotor tone in response to ₁-adrenoceptor stimulation. Such oscillations may represent a digitization of the Ca²⁺ signal, allowing a frequency-dependent control of the contractile response.⁵³ Frequency-dependent signaling would provide a mechanism to "fine tune" the contractile response. Alternatively, oscillations may allow Ca²⁺ to function as a second messenger while obviating the adverse effects of a sustained elevation in [Ca²⁺]_i.³¹ Such a sustained elevation could desensitize Ca²⁺-sensitive cellular response elements and/or increase energy loss due to stimulation of Ca²⁺-activated ATP-dependent enzymes. Because the oscillations do not depend on Ca²⁺ influx via VOCs, it is likely that PASMCs are not membrane oscillators but that they represent cytosolic oscillators.⁵³ The oscillations, at least in part, could be explained by periodic formation of IP₃-triggering bursts of Ca²⁺ release.⁵⁴ However, a negative-feedback loop involving PKC does not appear to be critically involved. Alternatively, IP₃ may be continuously present in the cell, and the action of IP₃ could be periodic by virtue of a negative-feedback loop operated through Ca²⁺ itself.⁵⁵ Transsarcolemmal Ca²⁺ influx via receptor-operated Ca²⁺ channels may be involved in priming or refilling the IP₃ Ca²⁺ store. Regardless of the precise mechanism of control, Ca²⁺ oscillations in PASMCs may be involved in the acute modulation of vasomotor tone in response to neural, humoral, or locally derived vasoactive factors.

Summary
In summary, the major finding of the present study is that ₁-adrenoceptor activation with PE triggers persistent Ca²⁺ oscillations in PASMCs via the phospholipase C signaling pathway. The oscillations are dependent on the presence of extracellular Ca²⁺ but do not require activation of voltage-operated Ca²⁺ channels. In addition, thapsigargin-sensitive and caffeine-sensitive, but not ryanodine-sensitive, Ca²⁺ stores appear to be involved. Protein phosphorylation by protein kinases is not involved in regulating the oscillations. The mechanisms that trigger and maintain these Ca²⁺ oscillations are complex and likely involve an interplay between multiple downstream cellular signals associated with Ca²⁺ regulation.

	Selected Abbreviations and Acronyms

 

DG = diacylglycerol IP3 = inositol 1,4,5-trisphosphate LB = loading buffer PASMC = pulmonary artery smooth muscle cell PE = phenylephrine PKC = protein kinase C SR = sarcoplasmic reticulum VOC = voltage-operated Ca2+ channel

	Acknowledgments

 
This study was supported by National Heart, Lung, and Blood Institute grants HL-38291 and HL-40361. Dr Hamada was also supported by Prof Osafumi Yuge, MD, PhD, Department of Anesthesiology, Hiroshima University School of Medicine, Hiroshima, Japan. The authors gratefully acknowledge the creative efforts of Peggy Kirian in developing the protocol for isolating freshly dispersed pulmonary arterial myocytes and her expertise in performing the electrophysiological recordings. The authors would also like to thank Ronnie Sanders for outstanding work in preparing this manuscript.

Received January 9, 1997; accepted July 18, 1997.

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