Propofol Attenuates Capacitative Calcium Entry in Pulmonary Artery Smooth Muscle Cells

Pulmonary arteries were isolated from adult mongrel dogs. The technique of euthanasia was approved by the Cleveland Clinic Institutional Animal Care and Use Committee (Cleveland, OH). 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, exsanguinated by controlled hemorrhage via  a femoral artery catheter, and euthanized with electrically induced ventricular fibrillation. A left lateral thoracotomy was performed, and the heart and lungs were removed en bloc . The pulmonary arteries were isolated and dissected in a laminar flow hood during sterile conditions.

Primary cultures of PASMCs were obtained as previously described. 11Intralobar pulmonary arteries (ID = 2–4 mm) were carefully dissected and prepared for tissue culture. Explant cultures were prepared according to the method of Campbell and Campbell, 12with 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-mm²pieces and explanted in 25-cm²culture dishes nourished by D-MEM/F-12 medium (Gibco, Grand Island, NY) containing 10% fetal bovine serum and a 1% antibiotic–antimycotic mixture solution (10,000 units/ml penicillin, 10,000 μg/ml streptomycin, 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 7–10 days before being subcultured nonenzymatically to 35-mm glass dishes designed for fluorescence microscopy (ΔT system; Bioptechs Inc., Butler, PA). Cells were used for experimentation within 72 h. Cells from the first and second passage were used for experiments. More than 90% of the cells stained positive for smooth muscle α actin. 11 

Pulmonary artery smooth muscle cells were loaded with fura 2 as previously described. 11Twenty-four hours before experimentation, the culture medium containing 10% fetal bovine serum was replaced with serum-free medium to arrest cell growth, 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. PASMCs were washed twice in loading buffer, which contained 125 mm NaCl, 5 mm KCl, 1.2 mm MgSO⁴, 11 mm glucose, 1.8 mm CaCl², 25 mm HEPES, and 0.2% bovine serum albumin, at pH 7.40 adjusted with NaOH. PASMCs were then incubated in loading buffer containing 2 μm fura 2/AM, the acetoxymethyl ester derivative of fura 2 (Molecular Probes, Eugene, OR), at ambient temperature for 30 min. After the 30-min loading period, the cells were washed twice in loading buffer and incubated 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. 11Culture 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 (Olympus America Inc., Lake Success, NY). Fluorescence measurements were obtained from either individual PASMCs or from a cluster (two to three cells) of neighboring cells 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 cells were superfused continuously at 1 ml/min with Krebs-Ringer buffer, which contained 125 mm NaCl, 5 mm KCl, 1.2 mm MgSO², 11 mm glucose, 2.5 mm CaCl², and 25 mm HEPES, 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 (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 PASMCs were continuously monitored at a sampling frequency of 25 Hz and were collected using a software package from Photon Technology International.

Capacitative Ca²⁺entry is triggered by the depletion of intracellular Ca²⁺stores. Thapsigargin increases [Ca²⁺]ⁱvia  irreversible inhibition of sarcoplasmic reticulum Ca²⁺–adenosine triphosphatase (ATPase). 13In the absence of extracellular Ca²⁺, thapsigargin (1 μm) was used to deplete sarcoplasmic reticulum Ca²⁺stores. With thapsigargin still present, capacitative Ca²⁺entry was then induced by restoring the extracellular Ca²⁺concentration (2.2 mm). 5The effects of propofol (1, 10, and 100 μm), tyrosine kinase inhibition (tyrphostin 23: 100 μm), PKC activation (phorbol 12-myristate 13-acetate: 1 μm) and PKC inhibition (bisindolylmaleimide: 1 μm) on capacitative Ca²⁺entry were assessed.

Propofol, thapsigargin, phorbol 12-myristate 13-acetate (all obtained from Research Biochemical International, Natick, MA), tyrphostin 23 (Calbiochem, La Jolla, CA), and bisindolylmaleimide 1 (Sigma, St. Louis, MO) were all dissolved in dimethyl sulfoxide as stock solutions. Aliquots of each stock solution were diluted 1:1000 in Krebs-Ringer buffer to achieve final concentrations in the bath. Similar dilutions of dimethyl sulfoxide in Krebs-Ringer buffer have no effect on [Ca²⁺]ⁱ. Pure propofol was used to avoid any effects of the intralipid emulsion on the fluorescence signal.

Peak and sustained increases in [Ca²⁺]ⁱwere measured in PASMCs 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 capacitative Ca²⁺entry response after thapsigargin pretreatment. This value was set at 100%. Therefore, each cell served as its own control. The “peak” response was calculated as the fluorescence change from baseline to peak fluorescence. The “sustained” response represents the fluorescence values measured 5 min after reintroduction of Ca²⁺to the buffer. Results are presented as mean ± SEM. Statistical analysis was performed using repeated-measures analysis of variance followed by Bonferroni–Dunn post hoc  testing. Differences were considered statistically significant at P < 0.05.

Capacitative Ca²⁺entry can be triggered by thapsigargin-induced depletion of intracellular Ca²⁺stores. In the absence of extracellular Ca²⁺, thapsigargin (1 μm) increased [Ca²⁺]ⁱby 182 ± 11%, followed by a return of [Ca²⁺]ⁱto baseline values (fig. 1, top). Once the baseline fluorescence signal had stabilized, the extracellular Ca²⁺concentration ([Ca²⁺]^o) was restored (2.2 mm) in the continued presence of thapsigargin (fig. 1, top). Restoring [Ca²⁺]^oresulted in a rapid peak increase (246 ± 12% of baseline) in [Ca²⁺]ⁱand a sustained increase (187 ± 7% of baseline) in [Ca²⁺]ⁱ(i.e. , capacitative Ca²⁺entry was induced). The sustained increase in [Ca²⁺]ⁱreturned to baseline when [Ca²⁺]^owas removed. The reproducibility of inducing capacitative Ca²⁺entry was assessed by sequentially removing and restoring [Ca²⁺]^othree consecutive times in the continued presence of thapsigargin. There were no significant differences in the peak or sustained increases in [Ca²⁺]ⁱwhen [Ca²⁺]^owas restored three consecutive times (fig. 1, bottom).

After depletion of sarcoplasmic reticulum Ca²⁺stores with thapsigargin, capacitative Ca²⁺entry was compared in the absence or presence of propofol, which was added to the superfusion buffer 5 min before restoring [Ca²⁺]^oa second time (fig. 2, top). Propofol had no effect on baseline [Ca²⁺]ⁱbefore the addition of [Ca²⁺]^o. Propofol caused dose-dependent decreases in both the peak and sustained increases in [Ca²⁺]ⁱwhen [Ca²⁺]^owas restored (fig. 2, bottom). After washout of propofol, capacitative Ca²⁺entry was similar in magnitude to the response measured before propofol administration (fig. 2, top).

We previously demonstrated that tyrosine kinases play a role in regulating capacitative Ca²⁺entry in PASMCs. 5In the current study, tyrphostin 23 (100 μm) was used to inhibit tyrosine kinases. Tyrosine kinase inhibition attenuated both the peak (67 ± 4% of control) and sustained (75 ± 5% of control) increases in [Ca²⁺]ⁱmediated through capacitative Ca²⁺entry (fig. 3). However, in the presence of tyrosine kinase inhibition, propofol (100 μm) further attenuated the peak (46 ± 4% of control) and sustained (55 ± 2% of control) increases in [Ca²⁺]ⁱwhen [Ca²⁺]^owas restored (fig. 4).

Protein kinase C has been implicated in the regulation of capacitative Ca²⁺entry in a variety of cell types. 14–16However, the extent to which PKC is involved in capacitative Ca²⁺entry in PASMCs has not been previously investigated. Activation of PKC with phorbol 12-myristate 13-acetate (1 μm) attenuated both the peak (48 ± 1% of control) and sustained (53 ± 3% of control) increases in [Ca²⁺]ⁱmediated via  capacitative Ca²⁺entry (fig. 5). In contrast, PKC inhibition with bisindolylmaleimide (1 μm) potentiated both the peak (132 ± 11% of control) and sustained (120 ± 4% of control) increases in [Ca²⁺]ⁱwhen [Ca²⁺]^owas restored (fig. 6). Moreover, in the presence of PKC inhibition, propofol (100 μm) had no effect on capacitative Ca²⁺entry (fig. 7). Thus, PKC inhibition abolished the propofol-induced attenuation in capacitative Ca²⁺entry.

The main findings of our study are as follows. First, both tyrosine kinases and PKC are involved in the signal transduction pathway for capacitative Ca²⁺entry in PASMCs. Tyrosine kinases positively regulate capacitative Ca²⁺entry, whereas PKC negatively regulates capacitative Ca²⁺entry. Second, propofol causes dose-dependent inhibition of capacitative Ca²⁺entry in PASMCs. Third, the propofol-induced attenuation of capacitative Ca²⁺entry is still observed after inhibition of tyrosine kinases. Finally, PKC inhibition abolishes the propofol-induced attenuation in capacitative Ca²⁺entry.

It is well known that [Ca²⁺]ⁱis an important determinant in the regulation of cardiac and smooth muscle contraction. In vascular smooth muscle, agonist-induced increases in [Ca²⁺]ⁱprimarily occur via  release of Ca²⁺from intracellular stores by a 1,4,5 inositol triphosphate–dependent mechanism. In addition, some agonists can trigger Ca²⁺influx across the sarcolemma, primarily via  voltage-gated or receptor-operated Ca²⁺channels. An increase in [Ca²⁺]ⁱactivates the myosin light chain kinase through a calmodulin-dependent mechanism, resulting in phosphorylation of the myosin light chains and initiation of contraction. 17[Ca²⁺]ⁱis ultimately restored either by pumping the Ca²⁺out of the cell via  the sarcolemmal Ca²⁺ATPase or Na⁺–Ca²⁺exchanger, or by resequestering Ca²⁺into the intracellular store by the sarcoplasmic reticulum Ca²⁺ATPase. 18,19 

In addition to the aforementioned sarcolemmal ion channels, Ca²⁺influx can also be controlled by the filling-state of the intracellular Ca²⁺store. The concept of capacitative Ca²⁺entry was first postulated by Putney. 1According to his model, depletion of intracellular Ca²⁺stores results in activation of a Ca²⁺influx pathway that is somehow sensitive to the state of filling of intracellular Ca²⁺stores. Influx of Ca²⁺via  capacitative Ca²⁺entry refills the intracellular Ca²⁺stores that have been depleted in response to agonist activation. In the current study, thapsigargin was used as a tool to deplete the sarcoplasmic reticulum pool of Ca²⁺in the absence of extracellular Ca²⁺and thereby activate capacitative Ca²⁺entry. Consistent with our previous study, 5the amplitude of the thapsigargin-induced increase in [Ca²⁺]ⁱwas variable, which likely reflects differences in the size of the sarcoplasmic reticulum Ca²⁺store in different cells. The size of the sarcoplasmic reticulum Ca²⁺store may depend on the cell passage number, the phase of the cell cycle, the length of time in serum-free medium, or whether the response was derived from an individual cell or a cluster of two to three neighboring cells. Restoring [Ca²⁺]^ostimulated capacitative Ca²⁺entry, which was typically characterized by both a peak and sustained increase in [Ca²⁺]ⁱ. The rapid peak increase in [Ca²⁺]ⁱresults from massive influx of Ca²⁺into the cytosol via  SK&F 96365–sensitive Ca²⁺channels that open in response to depletion of sarcoplasmic reticulum Ca²⁺stores. 5The sustained or prolonged increase in [Ca²⁺]ⁱis more complex. Because thapsigargin is an irreversible inhibitor of the sarcoplasmic reticulum Ca²⁺ATPase, the sarcoplasmic reticulum is incapable of refilling with Ca²⁺despite the increased availability of cytosolic free Ca²⁺. As a result, capacitative Ca²⁺entry across the sarcolemma is sustained, and a new steady state level of [Ca²⁺]ⁱis achieved as other mechanisms regulating Ca²⁺extrusion (Na⁺–Ca²⁺exchanger and the sarcolemmal Ca²⁺ATPase) begin to offset the continued influx of Ca²⁺. If the sarcoplasmic reticulum is allowed to refill, as we have previously demonstrated using a reversible inhibitor of the sarcoplasmic reticulum Ca²⁺ATPase (cyclopiazonic acid), the response to capacitative Ca²⁺entry is only transient, and a sustained phase is not evident. 5 

Thapsigargin-induced capacitative Ca²⁺entry does not involve activation of intracellular second messengers (e.g. , 1,4,5 inositol triphosphate). The cellular mechanism that mediates capacitative Ca²⁺entry has been intensively investigated, although it has yet to be definitively identified. Various models have been postulated to explain how the sarcoplasmic reticulum communicates with the plasma membrane. These models can generally be divided into those that propose the existence of a diffusible factor and those that suggest that the signal is transferred via  protein phosphorylation and dephosphorylation. 2Potentially important diffusible second messengers released from the storage organelles include cytochrome P450 metabolites, 20G proteins, 21or a low-molecular-weight compound called Ca²⁺influx factor. 22Models based on phosphorylation and dephosphorylation interactions suggest that PKC activation either inhibits 14,23or stimulates 24capacitative Ca²⁺entry depending on the cell type, whereas tyrosine kinase or protein kinase A activation are consistently associated with activation of capacitative Ca²⁺entry. 14,25–27 

Because capacitative Ca²⁺entry is involved in the regulation of [Ca²⁺]ⁱand vasomotor tone in PASMCs, 5the ion channel associated with capacitative Ca²⁺entry may serve as a cellular target for propofol. In cultured A10 and rat aortic smooth muscle cells, propofol was reported to inhibit voltage-gated Ca²⁺channels but to have no effect on capacitative Ca²⁺entry. 6In contrast, propofol attenuated capacitative Ca²⁺entry in cultured aortic smooth muscle cells from normotensive rats, 28and this effect was even more prominent in hypertensive rats. In our study using canine PASMCs, propofol caused a dose-dependent decrease in capacitative Ca²⁺entry. Typical free plasma concentrations of propofol range from 1 to 10 μm. Thus, propofol attenuated capacitative Ca²⁺entry at clinically relevant concentrations. Tyrosine kinase inhibition also attenuated capacitative Ca²⁺entry. However, in the presence of tyrosine kinase inhibition with tyrphostin 23, propofol further suppressed capacitative Ca²⁺entry. These results suggest that inhibition of tyrosine kinases is not the primary mechanism for propofol-induced inhibition of capacitative Ca²⁺entry. In contrast, inhibition of PKC with bisindolylmaleimide resulted in potentiation of capacitative Ca²⁺entry, whereas activation of PKC with phorbol 12-myristate 13-acetate resulted in attenuation of capacitative Ca²⁺entry. Moreover, pretreatment with bisindolylmaleimide prevented the propofol-induced attenuation of capacitative Ca²⁺entry. Taken together, these results suggest that propofol inhibits capacitative Ca²⁺entry via  a PKC-dependent mechanism. The cellular target for this propofol-induced, PKC-mediated attenuation of capacitative Ca²⁺entry remains to be elucidated. The propofol-induced attenuation of the peak response may either directly or indirectly be caused by PKC-dependent inhibition of Ca²⁺channels that mediate capacitative Ca²⁺entry, whereas attenuation of the sustained response may involve effects on the Na⁺–Ca²⁺exchanger or the sarcolemmal Ca²⁺ATPase. The current results are consistent with previous reports that propofol activates purified brain PKC. 29,30Moreover, we have preliminary data that suggest that propofol increases myofilament Ca²⁺sensitivity in PASMCs via  a PKC-dependent mechanism. 31 

In previous studies from our laboratory, 5,11we demonstrated that α-adrenoreceptor–mediated Ca²⁺oscillations were not altered by the nonselective, broad-range protein kinase inhibitor staurosporine. In contrast, the Ca²⁺oscillations were abolished by SK&F 96365, an inhibitor of capacitative calcium entry, and were attenuated by tyrosine kinase inhibition with genestein or tyrphostin. In this study, selective inhibition of PKC potentiated capacitative Ca²⁺entry, whereas selective inhibition of tyrosine kinases attenuated capacitative Ca²⁺entry. Because staurosporine effectively blocks both tyrosine kinases and PKC, the lack of effect of staurosporine on the α-adrenoreceptor–mediated Ca²⁺oscillations is likely a result of offsetting effects on capacitative Ca²⁺entry, resulting in no net effect on the Ca²⁺oscillations. Although propofol (1–10 μm) attenuated capacitative Ca²⁺entry in this study, we previously reported that the propofol-induced attenuation of α-adrenoreceptor–mediated Ca²⁺oscillations was apparent only at supraclinical concentrations. 9It should be noted that capacitative Ca²⁺entry is not the only mechanism regulating α-adrenoreceptor–mediated Ca²⁺oscillations. Moreover, regulation of capacitative Ca²⁺entry and Ca²⁺oscillations has not been entirely elucidated but appears to involve multiple mechanisms. In the current study, we demonstrated opposing actions of tyrosine kinases and PKC activation on capacitative Ca²⁺entry (CCE). It is possible that propofol alters multiple mechanisms involved in the regulation of the Ca²⁺oscillations, some of which may offset the effects of the other.

Although our results suggest that the effects of propofol on capacitative Ca²⁺entry are primarily mediated via  activation of PKC, alternative interpretations are possible. Propofol alone and tyrphostin alone inhibited capacitative Ca²⁺entry by approximately 30–35%. In the setting of tyrosine kinase inhibition, propofol further attenuated capacitative Ca²⁺entry by approximately 20%. Therefore, it could be argued that a portion of the inhibitory effect of propofol on capacitative Ca²⁺entry may be mediated via  inhibition of tyrosine kinases. However, this possibility seems unlikely because if the inhibitory effect of propofol on CCE is mediated by a pathway different from PKC, then propofol should continue to exert an inhibitory effect on CCE in the presence of PKC inhibition. Our results indicate that it did not. Given that PKC activation attenuated CCE and PKC inhibition abolished the propofol-induced attenuation in CCE, it seems reasonable to conclude that the effects of propofol are mediated (at least primarily) through PKC activation. This PKC-mediated attenuation of capacitative Ca²⁺entry induced by propofol does not appear to be a general characteristic of intravenous anesthetics. In preliminary studies, 32thiopental had no effect on capacitative Ca²⁺entry, whereas ketamine attenuated capacitative Ca²⁺entry via  a mechanism that did not involve PKC. These results indicate that intravenous anesthetics can have differential effects on capacitative Ca²⁺entry that are mediated by more than one cellular mechanism.

In summary, propofol attenuates capacitative Ca²⁺entry in PASMCs. This effect is not altered by inhibition of tyrosine kinases but is abolished by inhibition of PKC. Capacitative Ca²⁺entry should be considered as a possible cellular target for anesthetic agents that alter vascular smooth muscle tone.