The authors investigated the effects of intravenous anesthetics on alpha-adrenergic-induced oscillations in intracellular free calcium concentration ([Ca2+]i) in individual pulmonary artery smooth muscle cells (PASMCs).
PASMCs were cultured from explants of canine intrapulmonary artery. Fura-2-loaded PASMCs were continuously superfused with phenylephrine (10 microM) at 37 degrees C on the stage of an inverted fluorescence microscope. Measurement of [Ca2+]i was via a dual wavelength spectrofluorometer. Intravenous anesthetics were added to the superfusate to assess their effects on the phenylephrine-induced [Ca2+]i oscillations.
Resting [Ca2+]i was 103 +/- 6 nM. Phenylephrine stimulated [Ca2+]i oscillations, reaching a peak concentration of 632 +/- 20 nM and a frequency of 1.53 +/- 0.14 transients/min. The effects of phenylephrine were dose-dependent. The effects of intravenous anesthetics on phenylephrine-induced [Ca2+]i oscillations were dose-dependent. Ketamine (100 microM) reduced the amplitude (221 +/- 22 nM) but not the frequency (1.48 +/- 0.11/min) of the oscillations, whereas thiopental (100 microM) decreased the amplitude (270 +/- 20 nM) and the frequency (1.04 +/- 0.10/min). Propofol (100 microM) and the Intralipid vehicle inhibited the amplitude (274 +/- 11 nM) but not the frequency (1.39 +/- 0.11/min) of the oscillations. The effects of ketamine and thiopental, but not propofol, were evident at clinically relevant concentrations.
Ketamine, thiopental, and propofol exerted differential effects to inhibit the amplitude or the frequency of phenylephrine-induced [Ca2+]i oscillations in individual PASMCs. Thus, intravenous anesthetics may alter the pulmonary vascular response to alpha-adrenoreceptor activation by directly inhibiting [Ca2+]i signaling in PASMCs.
In the pulmonary circulation, in vitro studies using isolated lungs or pulmonary arterial rings have generally demonstrated that intravenous anesthetics exert a vasodilator influence and attenuate agonist-induced increases in vasomotor tone. [1–4] Although steady state vasomotor tone is primarily regulated by K sup + channels, [5,6] agonists that increase vascular smooth muscle intracellular free calcium concentration [Ca2+]iare important for the acute modulation of vasomotor tone in response to neural, humoral, or local activation. We recently reported that sympathetic alpha-adrenergic stimulation of individual canine pulmonary artery smooth muscle cells (PASMCs) induced oscillations in [Ca2+]i. * RF 7* Changes in the amplitude or frequency of [Ca2+]ioscillations could play a role in the acute modulation of pulmonary vasomotor tone in response to agonist activation. The effects of intravenous anesthetics on agonist-induced [Ca2+]isignaling in PASMCs have not been examined. The goal of the present study was to investigate the effects of three intravenous anesthetics (ketamine, thiopental, and propofol) on sympathetic alpha-adrenergic-induced [Ca2+]ioscillations in individual PASMCs. We tested the hypothesis that intravenous anesthetics would reduce the amplitude or frequency of [Ca2+]ioscillations in response to the alpha-agonist, phenylephrine. Our results indicate that intravenous anesthetics exert differential effects to directly inhibit [Ca2+]isignaling in response to agonist-induced activation in individual PASMCs.
Materials and Methods
Pulmonary arteries were isolated from adult male mongrel dogs. The technique of euthanasia was approved by the Institutional Animal Care and Use Committee. All procedures were performed aseptically during general anesthesia (fentanyl, 20 micro gram/kg; pentobarbital sodium, 30 mg/kg, intravenous) with endotracheal intubation and positive pressure ventilation. A catheter was placed in the right femoral artery; the mobilizable blood volume was removed, and 30 ml of saturated KCl was administered intravenously. A left thoracotomy was performed through the fifth intercostal space. The heart and lungs were removed en bloc, and the pulmonary arteries were isolated and dissected in a laminar flow hood during sterile conditions.
Primary cultures of smooth muscle cells were obtained from segmental and subsegmental intralobar branches of pulmonary artery (the third and fourth generation of branches from the main pulmonary artery) having diameters less than 4 mm. Explant cultures were prepared according to the method of Campbell  with minor modifications. The endothelium was removed by gentle rubbing with a sterile cotton swab. The tunica adventitia was carefully removed together with the most superficial part of the tunica media. The remaining tunica media was cut into 2-mm2pieces that were explanted in 25-cm2culture flasks. The explants were nourished by D-MEM/F-12 medium (Gibco, Grand Island, NY) containing 10% fetal bovine serum, and 1% antibiotic-antimycotic mixture solution (10,000 U/ml of penicillin, 10,000 micro gram/ml of streptomycin, 25 micro gram/ml of amphotericin B) and kept in a humidified atmosphere of 5% CO2:95% air at 37 [degree sign] Celsius. PASMCs began to proliferate from explants after 7 days in culture. Cells were allowed to proliferate for an additional 7–10 days until subconfluence was achieved. Cells were then subcultured non-enzymatically to 35-mm culture dishes specially designed for fluorescence microscopy (Bioptechs Inc., Delta T system, Butler, PA). PASMCs were used for experimentation within 72 h after subculture. Cells from the first to third passage were used for experiments. These cells were routinely identified as smooth muscle cells using a fluorescein-labeled antibody directed at smooth muscle alpha actin.
Fura-2 Loading Procedure
Twenty-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 proteins in the medium.  PASMCs were washed twice in loading buffer, which contained (in mM): 125 NaCl; 5 KCl; 1.2 MgSO4; 11 glucose; 1.8 CaCl2; 25 HEPES; plus 0.2% bovine serum albumin, at pH 7.4 adjusted with NaOH. PASMCs were then incubated in loading buffer containing 2 micro Meter fura-2/AM, the acetoxymethyl 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 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. 
Determination of [Ca sup 2+] sub i
Culture dishes containing fura-2-loaded PASMCs were placed in a temperature regulated (37 [degree sign] Celsius) chamber (Bioptechs, Inc.) mounted on the stage of an Olympus IX-70 inverted fluorescence microscope (Olympus America Inc., Lake Success, NY). Fluorescence measurements were performed on individual smooth muscle cells on a cultured monolayer using a dual wavelength spectrofluorometer (Deltascan RFK6002, Photon Technology International, S. Brunswick, 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 (in mM): 125 NaCl; 5 KCl; 1.2 MgSO4; 11 glucose; 2.5 CaCl2; 25 HEPES at pH 7.4 adjusted with NaOH. The temperature of all solutions was maintained at 37 [degree sign] Celsius in a water bath. 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 single PASMCs were continuously monitored at a sampling frequency of 25 Hz and were collected using a software package from Photon Technology International (Felix[trademark symbol]).
Calibration of fura-2 can be performed either in situ or in cell-free conditions using fura-2 (free acid) in buffers with known Ca sup 2+ concentrations. Photobleaching and photochemical formation of fluorescent Ca2+-insensitive forms is a general problem with both methods of calibration. There are several additional problems associated with in situ methods :(1) incomplete hydrolysis of fura-2 acetoxymethylester, resulting in Ca2+ insensitive but fluorescent compounds;(2) sequestration of fura-2 in noncytoplasmic compartments;(3) dye loss; and (4) shifts in excitation emission spectra and the dissociation constant (Kd) for Ca2+ resulting from changes in ionic strength and viscosity. For these reasons, we decided not to quantify the fura-2 fluorescence ratio using in situ methods, but rather to use the fluorescence ratio as a qualitative indicator of changes in [Ca2+]i. Estimates of [Ca2+]iwere made by comparing the cellular fluorescence ratio with ratios acquired using fura-2 (free acid) in buffers containing known Ca2+ concentrations. [Ca2+] sub i was then calculated as described by Grynkiewicz et al. 
Pulmonary artery smooth muscle cells were pretreated with the beta-adrenoreceptor antagonist, propranolol (5 micro Meter), to eliminate any beta-agonist effect of phenylephrine. The effects of increasing doses of phenylephrine on the amplitude and frequency of [Ca sup 2+]iwere first assessed. In subsequent experiments, after establishing steady-state [Ca2+]ioscillations induced by 10 micro Meter phenylephrine, an intravenous anesthetic (ketamine, thiopental, or propofol) was added to the superfusate. Any given PASMC was exposed to only one anesthetic agent. Ketamine (100 mg/ml stock) and thiopental (50 mM stock) were administered at concentrations ranging from 3–1,000 micro Meter, with 5-min intervals between doses. Solution changes were accomplished by rapidly aspirating the buffer in the dish and transiently increasing the flow rate to 10 ml/min. Propofol was applied in its commercially available 10% Intralipid[registered sign] emulsion (10 mg/ml, propofol; 10% soybean oil; 2.25% glycerol; 1.2% egg lecithin) in a similar fashion over the same dose range (3–1,000 micro Meter). In a separate series of experiments, Intralipid[registered sign](10%) was added to the superfusate as a vehicle control for propofol at concentrations corresponding to those used in the propofol protocol.
The amplitude and frequency of phenylephrine-induced [Ca2+]ioscillations were measured in individual PASMCs. The amplitude was calculated by averaging the peak ratio obtained for four or five oscillations before and after each intervention. The change in the 340:380 fluorescence ratio was then calculated by subtracting the resting ratio value (baseline). 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. Data are expressed as the mean +/- SEM. Statistical analysis was performed using repeated measures analysis of variance (ANOVA) followed by Bonferroni/Dunn post hoc testing. Differences were considered statistically significant at P < 0.05.
Phenylephrine, propranolol, and thiopental were purchased from Sigma Chemical Co. (St. Louis, MO). Propofol (Diprivan[registered sign]) was obtained from Zeneca Pharmaceuticals (Wilmington, DE); Intralipid[registered sign] was obtained from Kabi Pharmacia (Clayton, NC), and ketamine (Ketalar[registered sign]) was obtained from Parke-Davis (Morris Plains, NJ).
Characteristics of Phenylephrine-induced [Ca sup 2+] sub i Oscillations in PASMCs
Resting values of [Ca2+]iwere 103 +/- 6 nM. A typical trace showing the dose-dependent effects of phenylephrine on the amplitude and frequency of [Ca2+]ioscillations in a single PASMC is shown in Figure 1(top). Summarized data depicting the dose-dependent effects of phenylephrine on the amplitude and frequency of [Ca2+]ioscillations are shown in Figure 1(bottom; n = 21 cells). The amplitude of the [Ca2+]ioscillations reached a plateau at a concentration of 10 micro Meter phenylephrine and achieved a peak [Ca2+]iof 632 +/- 20 nM. The frequency of phenylephrine-induced [Ca2+]ioscillations was also dose-dependent, but did not reach a plateau even at 100 micro Meter phenylephrine. The frequency of [Ca2+]ioscillations at 10 micro Meter phenylephrine was 1.53 +/- 0.14 transients/min. Tachyphylaxis to phenylephrine was never observed, and oscillations maintained a constant amplitude and frequency for more than 30 min. The frequency and amplitude of phenylephrine-induced [Ca2+]ioscillations were similar in cells through the third passage.
Effects of Ketamine on Phenylephrine-induced [Ca sup 2+] sub i Oscillations in PASMCs
(Figure 2) shows a typical trace demonstrating the dose-dependent inhibitory effects of ketamine (10–1000 micro Meter) on [Ca2+]ioscillations induced by phenylephrine (10 micro Meter) in a single PASMC. Figure 3summarizes the dose-dependent effects of ketamine on the amplitude and frequency of phenylephrine-induced [Ca2+]ioscillations. Ketamine (100 micro Meter) significantly reduced (P < 0.05) the amplitude (221 +/- 22 nM) but not the frequency (1.48 +/- 0.11/min) of [Ca2+]ioscillations induced by phenylephrine (n = 23 cells). Higher concentrations of ketamine (1000 micro Meter) resulted in a significant (P < 0.05) decrease in the frequency (1.16 +/- 0.09/min) of [Ca2+]ioscillations. The inhibitory effect of ketamine on the amplitude of phenylephrine-induced [Ca2+]ioscillations was significant at clinically relevant concentrations (shaded bar in Figure 3).
Effect of Thiopental on Phenylephrine-induced [Ca sup 2+] sub i Oscillations in PASMCs
(Figure 4) summarizes the dose-dependent effects of thiopental on [Ca2+]ioscillations induced by phenylephrine (10 micro Meter). Thiopental (100 micro Meter) significantly decreased (P < 0.05) the amplitude (270 +/- 20 nM) and the frequency (1.04 +/- 0.10/min) of [Ca2+]ioscillations. The inhibitory effects of thiopental on the amplitude and frequency of the [Ca2+]ioscillations were dose-dependent and significant at clinically relevant concentrations (n = 22 cells).
Effects of Propofol and Intralipid[registered sign] on Phenylephrine-induced [Ca sup 2+] sub i Oscillations in PASMCs
The dose-dependent effects of propofol and Intralipid[registered sign] on the amplitude and frequency of [Ca2+]ioscillations induced by phenylephrine (10 micro Meter) are summarized in Figure 5. Propofol (100 micro Meter) exerted a significant inhibitory effect (P < 0.05) on the amplitude (274 +/- 11 nM) but not the frequency (1.39 +/- 0.11/min) of [Ca2+]ioscillations. Supratherapeutic concentrations of propofol (1000 micro Meter) caused a small but significant (P < 0.05) attenuation in the frequency (1.17 +/- 0.08/min) of [Ca2+]ioscillations. The inhibitory effect of propofol on the amplitude of [Ca2+]ioscillations was dose-dependent (n = 19 cells).
Similar to propofol, the Intralipid[registered sign] vehicle attenuated the amplitude of the [Ca2+]ioscillations, with minimal effect on the frequency (Figure 5; n = 14 cells). Intralipid[registered sign](equivalent to 100 micro Meter propofol) inhibited (P < 0.05) the amplitude (282 +/- 9 nM) but not the frequency (1.42 +/- 0.10/min) of [Ca2+]ioscillations. Supratherapeutic concentrations of Intralipid[registered sign](equivalent to 1,000 micro Meter propofol) slightly decreased (P < 0.05) the frequency (1.30 +/- 0.09/min) of [Ca2+]ioscillations. At clinically relevant concentrations, neither propofol nor Intralipid[registered sign] had a significant effect on the amplitude or frequency of phenylephrine-induced [Ca2+]ioscillations in PASMCs.
The present study clearly shows that intravenous anesthetics exert dose-dependent inhibitory effects on the amplitude and frequency of phenylephrine-induced [Ca2+]ioscillations in individual PASMCs. The effects of propofol and the Intralipid[registered sign] vehicle on phenylephrine-induced [Ca2+]ioscillations were apparent only at concentrations higher than the clinically relevant range. In contrast, ketamine and thiopental inhibited the phenylephrine-induced [Ca2+]ioscillations in a differential manner at clinically relevant concentrations. Ketamine reduced the amplitude of the [Ca2+]ioscillations, whereas thiopental inhibited the amplitude and the frequency of [Ca2+]ioscillations.
Signal Transduction Pathway for Phenylephrine-induced [Ca sup 2+] sub i Oscillations in PASMCs
Sympathetic alpha-adrenoreceptors are G-protein-coupled receptors that mediate vascular smooth muscle contraction by increasing [Ca2+]i.  Agonist-induced stimulation of alpha-adrenoreceptors results in the activation of phospholipase C, which hydrolyzes polyphosphatidylinositol 4,5 bisphosphate (PIP2) to inositol 1,4,5 triphosphate (IP3) and diacylglycerol.  IP sub 3 can increase [Ca2+]ivia the release of [Ca2+] from intracellular stores. Diacylglycerol stimulates protein kinase C,  which may be involved in the maintenance of sustained contractions. Oscillations in [Ca2+]iin response to agonist activation have been reported in several smooth muscle types, [16,17] which suggests that Ca2+ could exert its effects on smooth muscle tone through a frequency-dependent mechanism. We have recently demonstrated that cultured and freshly dispersed individual PASMCs exhibit [Ca2+]ioscillations in response to alpha-adrenoreceptor activation with phenylephrine.  These oscillations require extracellular Ca2+, are independent of voltage-gated Ca2+ channels, depend on phospholipase C activation, do not primarily involve protein kinase activation, and appear to involve the release of Ca2+ from intracellular stores that are sensitive to caffeine.  The frequency of [Ca2+]ioscillations may represent a digitization of the Ca2+ signal, allowing a frequency-dependent control of the contractile response.  Moreover, oscillations may permit Ca2+ to serve as a second messenger while avoiding the adverse effects of a sustained increase in [Ca2+]i,  which could desensitize Ca2+-sensitive cellular response elements or increase energy loss as a result of stimulation of Ca2+-activated ATP-dependent enzymes.
Ketamine Inhibits the Amplitude But Not the Frequency of Phenylephrine-induced [Ca sup 2+] sub i Oscillations in PASMCs
In vitro studies have demonstrated that ketamine directly inhibits vascular smooth muscle tone. [20,21] Ketamine attenuates the contractile responses to vasoactive agents and causes concentration-dependent relaxation of precontracted vascular smooth muscle preparations. [20–23] The mechanism responsible for the ketamine-induced attenuation of agonist-mediated contraction has been postulated to involve interference with transmembrane Ca2+ influx, the decreased release of Ca2+ from intracellular stores, or decreased availability of Ca2+ to the contractile apparatus. [20–23] It is unlikely that ketamine acts intracellularly on pathways downstream from IP3or the ryanodine receptor because it failed to inhibit contractions induced by IP3or caffeine in permeabilized rabbit ear artery.  Ketamine could have an effect on alpha-adrenoreceptors, receptor-operated Ca2+ channels, activation of phospholipase C, or IP3production.
Five minutes after induction with 2 mg/kg of ketamine, the plasma concentration reaches 60 micro Meter, and is maintained at 10 micro Meter during steady-state anesthesia.  Serum protein binding of ketamine is only 20%.  The free drug concentration 5 min after induction may reach 40–50 micro Meter, and higher plasma concentrations are likely 1–2 min after induction. Thus, our results suggest that clinically relevant concentrations of ketamine inhibit the amplitude of phenylephrine-induced [Ca2+]ioscillations in PASMCs.
Thiopental Inhibits the Amplitude and the Frequency of Phenylephrine-induced [Ca sup 2+] sub i Oscillations in PASMCs
Thiopental inhibited thromboxane-induced pulmonary vasoconstriction in isolated rabbit lungs  and endothelin-mediated contraction of porcine cerebral arteries.  Vasorelaxant effects of thiopental have been demonstrated in precontracted rat aorta  and canine cerebral and mesenteric arteries,  but not in pulmonary arterial rings. 
Inhibition of Ca2+ influx by thiopental has been demonstrated in rabbit lung  and in tracheal smooth muscle cells.  The inhibitory effect of thiopental could involve a decrease in Ca2+ release from the sarcoplasmic reticulum because it has been shown that thiobarbiturates inhibit caffeine-induced contraction in intact and chemically skinned canine mesenteric arteries. [4,31] These results suggest that thiopental could have extracellular and intracellular sites of action that regulate the amplitude and frequency of [Ca2+]ioscillations.
Peak serum concentrations of thiopental immediately after induction reach 100 micro gram/ml and decrease to 10 micro gram/ml by 15 min.  Because a large fraction ([approximately] 75%) of thiopental is bound to albumin in the blood, [32–34] the actual concentration of the free drug could range from 2.5 to 25 micro gram/ml, which corresponds to a serum concentration of 5–50 micro Meter. Therefore, our results indicate that clinically relevant concentrations of thiopental inhibit the amplitude and frequency of phenylephrine-induced [Ca2+]ioscillations in PASMCs.
Propofol and Intralipid[registered sign] Inhibit Phenylephrine-induced [Ca sup 2+] sub i Oscillations in PASMCs at Supratherapeutic Concentrations
Propofol is a widely used intravenous induction agent because of its rapid onset, short duration of action, and rapid elimination.  The effects of propofol on the pulmonary circulation are controversial. In humans, propofol has been reported to cause a transient increase in pulmonary vascular resistance immediately after induction.  In contrast, propofol has been reported to cause pulmonary vasodilation in humans with artificial hearts.  In pentobarbital-anesthetized dogs, propofol had no effect on either baseline pulmonary vasomotor tone or the magnitude of hypoxic pulmonary vasoconstriction.  In preliminary studies in chronically instrumented dogs, we observed that the effects of propofol on the pulmonary vascular pressure-flow relationship were tone-dependent.  Compared with the conscious state, propofol had no effect on the pulmonary circulation at baseline, whereas propofol exerted a marked vasoconstrictor effect when vasomotor tone was pharmacologically increased with a thromboxane mimetic.  In contrast, propofol has been demonstrated to cause direct and potent vasorelaxation in isolated rat pulmonary arterial rings. 
The cellular mechanism of action of propofol on phenylephrine-induced [Ca2+]ioscillations in PASMCs is unknown, although the inhibitory effect appeared to be entirely a result of the Intralipid[registered sign] vehicle. Supratherapeutic concentrations of propofol have been shown to inhibit voltage-gated Ca sup 2+ currents in porcine tracheal smooth muscle cells.  Because the phenylephrine-induced [Ca2+]ioscillations require extracellular Ca2+, propofol could inhibit the influx of extracellular Ca2+ via receptor-operated Ca2+ channels. Alternatively, the inhibitory effect of propofol could involve alterations in alpha-adrenoceptor function or more distal points in the signal transduction pathway for phenylephrine-induced [Ca2+]ioscillations.
The concentrations of propofol that caused inhibitory effects on phenylephrine-induced [Ca2+]ioscillations were almost certainly higher than those normally encountered in clinical practice. Typical serum concentrations for propofol range from 1 to 10 micro gram/ml, which is equivalent to 5–50 micro Meter. [40,41] However, the free drug concentration is much lower as a result of substantial serum protein binding. [42,43] Thus, the inhibitory effects of propofol in this study were only apparent at supratherapeutic concentrations.
The present study demonstrates that ketamine, thiopental, and propofol have differential effects on phenylephrine-induced [Ca2+] sub i oscillations in PASMCs. The precise cellular mechanisms that mediate the effects of these intravenous anesthetics on Ca2+ signaling in PASMCs remain to be elucidated.
The authors thank Ronnie Sanders for outstanding work in preparing this manuscript.