Intravenous Anesthetics Inhibit Capacitative Calcium Entry in Pulmonary Venous Smooth Muscle Cells

Pulmonary veins were isolated from adult mongrel dogs. The technique of euthanasia was approved by the Cleveland Clinic Institutional Animal Care and Use Committee (Cleveland, Ohio). All steps were performed aseptically during general anesthesia with intravenous pentobarbital sodium (30 mg/kg) and intravenous fentanyl citrate (20 μg/kg). The dogs were intubated and ventilated. After administration of heparin (6,000 U), the dogs were exsanguinated by controlled hemorrhage via  a femoral artery catheter and killed with electrically induced ventricular fibrillation. A left lateral thoracotomy was performed, and the heart and lungs were removed en bloc . The pulmonary veins ware isolated and dissected in a laminar flow hood using sterile procedures.

Primary cultures of PVSMCs were obtained from segmental and subsegmental branches of pulmonary vein (the third and fourth generations having diameters < 4 mm). The intralobar veins were carefully dissected and prepared for tissue culture. Explant cultures were prepared according to the method of Campbell and Campbell,15with 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-mm²pieces that were explanted in 25-cm²culture flasks. The explants were nourished by Dulbecco's modified Eagle medium/F-12 containing 10% fetal bovine serum and 1% antibiotic mixture solution (10,000 U/ml penicillin and 10,000 μg/ml streptomycin) and kept in a humidified atmosphere of 5% CO²–95% air at 37°C. PVSMCs began to proliferate from explants after 7 days in culture. Cells were allowed to grow for an additional 10–14 days until subconfluence was achieved. Cells were then subcultured to 35-mm glass dishes specially designed for fluorescence microscopy (Bioptechs ΔT system; Butler, PA). Cells from the first passage were used for experiments. The cells exhibited morphologic characteristics of vascular smooth muscle and expressed α-actin as assessed by Western blot analysis.

Twenty-four hours before experimentation, the culture medium containing 10% fetal bovine serum was replaced with serum-free medium to arrest cell growth, to allow for establishment of steady state cellular events independent of cell division, and to prevent a false estimate of [Ca²⁺]ⁱresulting from binding of available dye to serum protein in the medium. PVSMCs were loaded with the acetoxymethyl ester form of fura-2 (fura-2 AM: 2 μm) at ambient temperature. After the 30-min loading period, the cells were washed twice in Krebs-Ringer's buffer, which contained 125 mm NaCl, 5 mm KCl, 1.2 mm MgSO⁴, 11 mm glucose, 2.5 mm CaCl², and 25 mm HEPES at pH 7.40 adjusted with NaOH at ambient temperature for an additional 20 min before initiating the study. This provided enough time to wash away any extracellular fura-2 AM and for intracellular esterases to cleave fura-2 AM into the active fura-2.

Intracellular Ca²⁺concentration was measured as previously described.6Culture dishes containing fura-2–loaded PVSMCs were placed in a temperature-regulated (37°C) chamber (Bioptechs, Inc., Butler, PA) mounted on the stage of an Olympus IX-70 inverted fluorescence microscope (Olympus America Inc., Lake Success, NY). Fluorescence measurements were obtained from individual PVSMCs in a culture monolayer using a dual-wavelength spectrofluorometer (Deltascan RFK6002; Photon Technology International, Lawrenceville, NJ) 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 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 superfusing it with a pipet. 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, 380, and 340/380 ratio) originating from PVSMCs were continuously monitored at a sampling frequency of 25 Hz and were collected using a software package from Photon Technology International.

In the absence of extracellular Ca²⁺, thapsigargin was used to deplete intracellular Ca²⁺stores. Thapsigargin is an irreversible sarcoplasmic reticulum Ca²⁺-adenosine triphosphatase inhibitor and can induce CCE.16After depletion of sarcoplasmic reticulum Ca²⁺stores, CCE was induced when extracellular Ca²⁺([Ca²⁺]^o, 2.2 mm) was restored. The effects of L-type voltage dependent Ca²⁺channel inhibition (verapamil, 10 μm), nonselective Ca²⁺channel inhibition (SKF 96365, 50 μm), TK inhibition (tyrphostin 23, 100 μm), PKC activation (phorbol 12-myristate 13-acetate, 1 μm), PKC inhibition (bisindolylmaleimide I, 1 μm), and ROK inhibition (Y27632, 10 μm) on CCE were investigated. The concentration of the inhibitors was chosen based on previous experience in PASMCs6,14and PVSMCs.12,13The effects of intravenous anesthetics (thiopental, 10–100 μm; midazolam, 10–100 μm; ketamine, 10–100 μm; and propofol, 10–100 μm), alone or in combination with a signaling pathway inhibitor, on CCE were assessed.

Verapamil, SKF 96365, tyrphostin 23, phorbol 12-myristate 13-acetate, bisindolylmaleimide (Sigma, St. Louis, MO), and propofol (Aldrich Chemical Co., Milwaukee, WI) were dissolved in dimethyl sulfoxide. The final chamber concentration of dimethyl sulfoxide was less than 0.1% (vol/vol). This diluent had no effect on CCE at the concentration used in these studies. Y27632 (Calbiochem, La Jolla, CA), ketamine (Fort Dodge Animal Health, Fort Dodge, IA), midazolam (American Pharmaceutical Partners Inc., Schaumburg, IL), and thiopental (Sigma) were dissolved in distilled water.

Data analysis was performed as previously described.14Peak and sustained increases in [Ca²⁺]ⁱwere measured in PVSMCs when the superfusion solution was switched from a Ca²⁺-free solution to a solution containing 2.2 mm Ca²⁺. Peak and sustained fluorescence ratio values were averaged before and after each intervention and are expressed as percent of control. The control response to which all interventions were compared was the first CCE response after thapsigargin pretreatment. This value was set at 100%. Therefore, each cell served as it own control. The peak response was calculated as the fluorescence change from baseline to peak fluorescence. The sustained response represents the fluorescence values measured when the 340/380 ratio was stable after reintroduction of Ca²⁺to the buffer. Results are presented as mean ± SEM. Statistical analysis was performed with analysis of variance and the Student t  test. Differences were considered statistically significant at P < 0.05.

To identify the presence of CCE in PVSMCs, thapsigargin was used to deplete sarcoplasmic reticulum Ca²⁺stores in the absence of extracellular Ca²⁺. Thapsigargin transiently increased [Ca²⁺]ⁱby 160 ± 6%, which gradually returned to baseline. Restoring extracellular Ca²⁺([Ca²⁺]^o, 2.2 mm) then caused a rapid peak increase in [Ca²⁺]ⁱ(155 ± 7% of baseline; P < 0.05), followed by a sustained increase in [Ca²⁺]ⁱ(129 ± 5% of baseline; P < 0.05), i.e. , CCE was stimulated in pulmonary venous smooth muscle cells (fig. 1A). The sustained increase in [Ca²⁺]ⁱreturned to baseline when [Ca²⁺]^owas removed. To identify the reproducibility of inducing CCE in the same PVSMC, [Ca²⁺]^owas sequentially restored and removed three consecutive times. There were no significant differences in the peak or sustained increases in [Ca²⁺]ⁱbetween the first and the second CCE, but the third CCE was slightly smaller in magnitude in the peak and sustained increases in [Ca²⁺]ⁱcompared with the first CCE (fig. 1B).

SKF 96365 is a nonselective Ca²⁺channel inhibitor that has been used by many investigators to inhibit CCE.17SKF 96365 (50 μm) was applied 5 min before [Ca²⁺]^owas restored the second time (fig. 2A). SKF 96365 attenuated both the peak and sustained increases in [Ca²⁺]ⁱdue to CCE (fig. 2B).

Verapamil was used to inhibit voltage-dependent Ca²⁺channels. Verapamil (10 μm) was applied 5 min before [Ca²⁺]^owas restored the second time. Verapamil had no effect on the peak or sustained increases in [Ca²⁺]ⁱdue to CCE (fig. 2B).

We previously demonstrated that TK plays a role in regulating CCE in PASMCs.6,14Tyrphostin 23 was used to inhibit TK. Tyrphostin 23 (100 μm) was applied 5 min before [Ca²⁺]^owas restored the second time (fig. 3A). Tyrphostin 23 attenuated both the peak and sustained increases in [Ca²⁺]ⁱdue to CCE (fig. 3B).

We previously demonstrated that PKC plays a role in regulating CCE in PASMCs.14Phorbol 12-myristate 13-acetate (1 μm) and bisindolylmaleimide I (1 μm) were used to activate and inhibit PKC, respectively. They were applied 5 min before [Ca²⁺]^owas restored the second time. Neither phorbol 12-myristate 13-acetate nor bisindolylmaleimide I had an effect on the peak or sustained increases in [Ca²⁺]ⁱdue to CCE (fig. 3B).

Y27632 was used to inhibit ROK. Y27632 (10 μm) was applied 5 min before [Ca²⁺]^owas restored the second time. Y27632 had no effect on the peak or sustained increases in [Ca²⁺]ⁱdue to CCE (fig. 3B).

The intravenous anesthetics were applied 15 min before [Ca²⁺]^owas restored the second time. Ketamine (10–100 μm) caused dose-dependent decreases in both the peak and sustained increases in [Ca²⁺]ⁱdue to CCE (fig. 4A). Thiopental (30–100 μm) caused dose-dependent decreases in both the peak and sustained increases in [Ca²⁺]ⁱdue to CCE (fig. 4B), although the lowest concentration of thiopental had no effect (fig. 4B). Midazolam (30–100 μm) caused dose-dependent decreases in both the peak and sustained increases in [Ca²⁺]ⁱdue to CCE (fig. 5A), whereas the lowest concentration of midazolam (10 μm) had no effect (fig. 5A). Propofol (100 μm) attenuated both the peak and sustained increases in [Ca²⁺]ⁱdue to CCE (fig. 5B), but lower concentrations of propofol (30 μm, 10 μm) had no effect (fig. 5B).

To determine whether the TK signaling pathway is involved in the anesthesia-induced attenuation of CCE, we investigated the effects of the anesthetics on CCE in the presence of TK inhibition. The intravenous anesthetics were applied before [Ca²⁺]^owas restored the second time. In the presence of tyrphostin 23, propofol further attenuated both the peak and sustained increases in [Ca²⁺]ⁱdue to CCE compared with TK inhibition alone (fig. 6). However, ketamine, thiopental, and midazolam had no effect on the peak or sustained increases in [Ca²⁺]ⁱdue to CCE in the presence of TK inhibition compared with TK inhibition alone (fig. 6).

Our results demonstrate that CCE exists in canine PVSMCs. The TK signaling pathway, but not the PKC and ROK pathways, is involved in CCE in canine PVSMCs. Clinically relevant concentrations of ketamine and thiopental attenuate CCE, whereas only supraclinical concentrations of midazolam and propofol have this effect. The TK signaling pathway is involved in the ketamine-, thiopental-, and midazolam-induced attenuation of CCE, whereas it is not involved in the propofol-induced attenuation of CCE.

Capacitative Ca²⁺entry has been demonstrated in a variety of cell types, including vascular smooth muscle cells.18,19In the current study, we used thapsigargin to deplete the sarcoplasmic reticulum pool of Ca²⁺in the absence of extracellular Ca²⁺and thereby activated CCE.20Restoring [Ca²⁺]^ocaused a rapid peak increase followed by a sustained increase in [Ca²⁺]ⁱ. This is the first demonstration that CCE exists in PVSMCs. When [Ca²⁺]ⁱwas restored and removed three times, there were no differences between the first and second CCE responses, but the third CCE was slightly reduced. Therefore, we assessed the effects of interventions by comparing the second CCE response to the first.

SKF 96365 has been used to block CCE after depletion of sarcoplasmic reticulum Ca²⁺stores in a variety of cell types.17In our study, SKF 96365 markedly attenuated both the peak and sustained increases in [Ca²⁺]ⁱdue to CCE, whereas verapamil had no effect. These results are consistent with the concept that CCE is insensitive to voltage-gated Ca²⁺channel inhibitors.

The basis of CCE is that release of Ca²⁺from intracellular stores increases Ca²⁺influx. The mechanism linking the decrease in intracellular Ca²⁺stores to the opening of plasma membrane Ca²⁺channels remains controversial. One theory postulates the release of a diffusible messenger by the pools, whereas other hypotheses involve a physical interaction between the empty stores and plasma membrane proteins, secretory vesicles, or even cytoskeletal elements.21It has been proposed that the sarcoplasmic reticulum might possess protein kinases or phosphatases capable of altering the phosphorylation state of ion channels.22Previous studies reported that PKC activation could inhibit23or facilitate24CCE. We have demonstrated that PKC negatively regulates CCE in PASMCs.14In the current study, neither the PKC activator, phorbol 12-myristate 13-acetate, nor the PKC inhibitor, bisindolylmaleimide I, had an effect on CCE in PVSMCs. We also assessed the role of another kinase, ROK, in CCE. We have recently reported that ROK is involved in agonist-induced pulmonary venous contraction12,13However, Y27632, a ROK inhibitor, had no effect on CCE in PVSMCs. This suggests that the ROK signaling pathway is not involved in CCE in PVSMCs.

It has been reported that depletion of intracellular Ca²⁺stores triggers tyrosine phosphorylation,25and inhibition of TK attenuates CCE in a number of cell types,26,27including smooth muscle.28We have demonstrated that inhibition of TK attenuates CCE in PASMCs.6,14In the current study, the TK inhibitor, tyrphostin 23, attenuated CCE in PVSMCs. This suggests that the TK signaling pathway is involved in CCE in PVSMCs.

It is well known that [Ca²⁺]ⁱplays an important role in the contraction of smooth muscle. The intravenous anesthetics ketamine,29propofol,30midazolam,31and thiopental30have been reported to inhibit smooth muscle contractile responses by reducing [Ca²⁺]ⁱ. Recently, we reported that ketamine attenuated acetylcholine-induced contraction in pulmonary veins.32Because CCE is involved in the regulation of [Ca²⁺]ⁱin PVSMCs, CCE may serve as a cellular target for intravenous anesthetics in PVSMCs. Therefore, we investigated the effects of intravenous anesthetics on CCE in PVSMCs. Our results indicate that clinical concentrations of ketamine33and thiopental34caused dose-dependent decreases in CCE in PVSMCs. Midazolam and propofol attenuated CCE only in supraclinical concentrations (midazolam: 100 μm, clinical concentration is 0.3–10 μm35; propofol: 100 μm, clinical concentration is 5–50 μm36). It has been reported that intravenous anesthetics differentially inhibit phenylephrine-induced [Ca²⁺]ⁱoscillations by inhibiting CCE in PASMCs.6,37Our results suggest that intravenous anesthetics may alter pulmonary venous tone by inhibiting CCE. To investigate the role of TK as a mechanism by which the anesthetics attenuated CCE, we performed experiments in PVSMCs after pretreatment with the TK inhibitor tyrphostin 23. Compared with TK inhibition alone, ketamine, thiopental, and midazolam no longer attenuated CCE after pretreatment with tyrphostin 23, suggesting that the TK signaling pathway is involved in the reductions in CCE caused by these anesthetics. In contrast, propofol continued to decrease CCE in the presence of tyrphostin 23, suggesting that inhibition of TK is not the primary mechanism for the propofol-induced inhibition of CCE. This result is consistent with a previous report from our laboratory that inhibition of TK is not the primary mechanism for propofol-induced inhibition of CCE in PASMCs.14We also reported that propofol attenuated CCE via  a PKC-dependent mechanism in PASMCs.14However, our current study demonstrated that PKC inhibition did not attenuate CCE in PVSMCs. Therefore, the propofol-induced attenuation of CCE in PVSMCs is not likely to involve PKC.

We acknowledge that results obtained from this in vitro  study can only be cautiously extrapolated to clinical practice. However, because pulmonary venous resistance is an important component of total pulmonary vascular resistance, our results provide new insight concerning the effect of intravenous anesthetics on pulmonary venous contraction.

In summary, CCE exists in PVSMCs. The TK signaling pathway positively regulates CCE, whereas the PKC and ROK signaling pathways are not involved. Ketamine, thiopental, and midazolam attenuate CCE via  a TK-dependent mechanism.