Inhibitory Effects of Propofol on Intracellular Signaling by Endothelin-1 in Aortic Smooth Muscle Cells 

Propofol was purchased from Aldrich (Tokyo, Japan).⁴⁵CaCl2 (10–40 mCi/mg), myo-[sup 3 H]inositol (81.5 Ci/mmol), [methyl-sup 3 H]choline chloride (85 Ci/mmol), and 3-[sup 125 I]iodotyrosyl endothelin-1 (2,000 Ci/mmol) were obtained from Amersham Japan (Tokyo, Japan). Endothelin-1 was purchased from Peptide Institute (Minoh, Japan). NaF was purchased from Sigma Chemical Company (St. Louis, MO). Other materials and chemicals were obtained from commercial sources. Propofol was dissolved in ethanol. The maximum concentration of ethanol in the culture medium was 0.1%, and this did not affect the measurement of⁴⁵Ca²+ influx, the formation of inositol phosphates and choline, or the binding assay. We used an assay buffer (consisting of 5 mM HEPES, pH 7.4; 150 mM NaCl; 5 mM KCl; 0.8 mM MgSO⁴; 1 mM CaCl sub 2; and 5.5 mM glucose) containing 0.01% bovine serum albumin (BSA) and 0.1% ethanol as the vehicle control for propofol. Endothelin-1 was dissolved in the assay buffer containing 0.01% BSA. We used the assay buffer containing 0.01% BSA as the vehicle control for endothelin-1.

A10 cells were obtained from the American Type Culture Collection (Rockville, MD). The cells (1 x 10⁵were seeded into 35-mm-diameter dishes and maintained at 37 [degree sign] Celsius in a humidified atmosphere of 5% carbon dioxide and 95% air in 2 ml Dulbecco's modified Eagle's medium containing 10% fetal calf serum. After 5 days, the medium was exchanged for 2 ml serum-free Dulbecco's modified Eagle's medium. The cells were used for experiments 48 h thereafter. To measure the formation of inositol phosphates, the medium was exchanged for 2 ml of inositol-free Dulbecco's modified Eagle's medium.

sup 45 Ca²+ influx was measured as previously described. [38]Briefly, the cultured cells were pretreated with various doses of propotol (0.1 micro Meter-0.1 mM) for 20 min in 1 ml of the assay buffer containing 0.01% BSA. The cells were stimulated by endothelin-1 in the presence of 5 micro Ci⁴⁵Ca²+ at 37 [degree sign] Celsius for the indicated periods. After four washes with 1 ml of the cold assay buffer containing [ethylenebis-(oxyethylenenitrilo)] tetraacetic acid, the reaction was immediately terminated by adding 1 ml 0.1% sodium dodecyl sulfate. The radioactivity of the lysate was then determined.

Cultured cells were labeled with myo-[sup 3 H]inositol (2 micro Ci/dish) for 48 h. The labeled cells were preincubated with 10 mM LiCl for 10 min at 37 [degree sign] Celsius in 1 ml of the assay buffer containing 0.01% BSA. The cells were stimulated by endothelin-1 or NaF at 37 [degree sign] Celsius. The reaction was terminated by adding 1 ml 30% trichloroacetic acid. The acidic supernatant was treated with diethyl ether to remove the acid and neutralized with 0.1 N NaOH. The supernatant was applied to an anion exchange column containing 1 ml Dowex AG1-X8 (100–200 mesh, formate form; Bio-Rad Laboratories, Hercules, CA). The radioactive inositol phosphates were eluted with 8 ml 0.1 M formic acid containing 1 M ammonium formate. [39,40]Pretreatment with propofol (0.1 micro Meter-0.1 mM) or vehicle was performed for 20 min.

To determine phosphatidylcholine-hydrolyzing phospholipase D activity in A10 cells, cultured cells were labeled with [methyl-sup 3 H]choline chloride (3 micro Ci/dish) for 72 h. The labeled cells were washed twice with 1 ml assay buffer. The cells were pretreated with propofol (0.1 micro Meter-0.1 mM) or vehicle for 20 min at 37 [degree sign] Celsius in 1 ml assay buffer containing 0.01% BSA, and then stimulated by endothelin-1. The reaction was terminated by adding 0.75 ml ice-cold methanol. The dishes were placed on ice for 30 min, and the contents were then transferred to tubes to which chloroform was added. They were then left standing on ice for 60 min. Chloroform and water were then added to give a final ratio of 1:1:0.9 (chloroform:methanol:water). The tubes were centrifuged at 14,000g for 5 min, and the upper aqueous methanolic phase was taken for analysis of the water-soluble choline-containing metabolites. Separation was conducted on a column containing 1 ml Dowex 50-WH sup +(200–400 mesh) as described [41]but with a minor modification. [42]We confirmed that standard [sup 3 H]choline, when applied to the column, can be eluted with 10 ml 1 M HCl. Briefly, the phase was diluted to 5 ml with water and applied to the column. Glycerophosphocholine and choline phosphate were removed with 24 ml water, and radioactive choline was eluted with 10 ml 1 M HCl.

Cultured cells were subjected to a binding assay that was essentially as described by Bronnegard et al., [43]with minor modifications. Briefly, the cells were pretreated with propofol (0.1 mM) or vehicle for 20 min at 37 [degree sign] Celsius in 1 ml 10 mM HEPES-buffered Dulbecco's modified Eagle's medium (pH 7.4). The cells were then incubated with various doses of [sup 125 I]endothelin-1 with or without a 1,000-fold molar excess of nonradioactive endothelin-1 for 20 min at 37 [degree sign] Celsius. At the end of the incubation, the cells were thoroughly washed with cold phosphate-buffered saline and solubilized using 1 ml 0.1% sodium dodecyl sulfate. The radioactivity of the lysate was then determined.

The radioactivity of⁴⁵Ca and³H samples was determined using a Beckman LS6500IC liquid scintillation spectrometer (Fullerton, CA). The radioactivity of¹²⁵I samples was determined using a Wallac 1480 WIZARD 3" automatic gamma counter (Turku, Finland).

The data were analyzed using one-way analysis of variance, followed by the Bonferroni correction for multiple comparisons between pairs. Probability values < 0.05 were considered significant. All data are presented as the mean +/- SD of triplicate determinations.

Endothelin-1 significantly increased⁴⁵Ca²+ influx from the extracellular space in a time-dependent manner (up to 10 min) in A10 cells (Figure 1(A)). The stimulatory effect of endothelin-1 on sup 45 Ca²+ influx was dose dependent over the range 0.1 nM-0.1 micro Meter (Figure 1(B)), with the maximum effect of endothelin-1 observed at 0.1 micro Meter.

Pretreatment with propofol, which did not affect⁴⁵Ca sup 2+ influx, attenuated the endothelin-1 (0.1 micro meter)-induced⁴⁵Ca²+ influx only at high concentrations (Figure 2). A significant effect of propofol was observed only at 0.1 mM, a dose that caused a 45% reduction in the effect of endothelin-1.

It has been reported that endothelin induces phospholipase C-catalyzed phosphoinositide hydrolysis in A10 cells. [31,35]We confirmed that endothelin-1 induced the formation of inositol phosphates in a time- and dose-dependent manner in these cells, with the maximum effect of endothelin-1 observed at 0.1 micro Meter (data not shown).

Pretreatment with propofol, which by itself had little effect on the formation of inositol phosphates, inhibited the endothelin-1 (0.1 micro Meter)-induced formation of inositol phosphates only at high concentrations (Figure 3and Figure 4). The inhibitory effect of propofol was dose dependent over the range 1 micro Meter-0.1 mM (Figure 4). The maximum effect of propofol was observed at 0.1 mM, a dose that caused a 70% reduction in the effect of endothelin-1.

Research has shown that ETA receptors are coupled to phosphoinositide-hydrolyzing phospholipase C via a pertussis toxin-insensitive GTP-binding protein in A10 cells. [31]For this reason, we examined the effect of propofol on the formation of inositol phosphates induced by NaF, an activator of heterotrimeric GTP-binding proteins. [33]The stimulatory effect of NaF on the formation of inositol phosphates was dose dependent for the range 1–40 mM, with the maximum effect observed at 40 mM (data not shown). In addition, we examined the effect of propofol on various doses of NaF-induced inositol phosphates formation. Propofol had no effect on the NaF (40 mM)-induced formation of inositol phosphates (Figure 4). Propofol also did not affect the inositol phosphates formation induced by doses of NaF < 40 mM (1,537 +/- 18 cpm for 10 mM NaF with pretreatment with vehicle; 1,596 +/- 259 cpm for 10 mM NaF with pretreatment with 0.1 mM propofol, as measured during a stimulation for 20 min).

It has been reported that endothelin-1 stimulates phosphatidylcholine-hydrolyzing phospholipase D in A10 cells. [36]Phosphatidylcholine hydrolysis by phospholipase D results in the formation of phosphatidic acid and choline. [21–23]We confirmed that endothelin-1 significantly increased the formation of choline in a time-dependent manner (up to 30 min) in these cells (Figure 5(A)). The stimulatory effect of endothelin-1 on choline formation was dose dependent for the range 1 nM-0.1 micro Meter, the maximum effect observed at 0.1 micro Meter (Figure 5(B)).

Only at high concentration (0.1 mM) did propofol significantly reduce the endothelin-1 (0.1 micro Meter)-induced choline formation, reducing it by 60%.

To identify the point at which propofol exerts its inhibitory effect on endothelin-1-induced intracellular signaling in A10 cells, we examined the effect of propofol on endothelin-1 binding to its specific receptor. It has been reported that Scatchard analysis indicates a single high-affinity population of endothelin-1 receptors with dissociation constant 0.12 nM in A10 cells. [37]We used doses of endothelin-1 of 50 and 150 pM. Propofol (0.1 mM) had no effect on the binding ability of endothelin-1 (Table 1).

In the present study, we showed that endothelin-1 induced a Ca²+ influx from the extracellular space in cultured A 10 cells. It has been reported that endothelin-1 stimulates phosphoinositide hydrolysis by phospholipase C in such cells, [31,35]and phosphoinositide hydrolysis by phospholipase C leads to the formation of inositol phosphates and diacylglycerol. [20,21]Inositol 1,4,5-trisphosphate serves as a messenger for mobilizing Ca²+ from intracellular stores, [20]and diacylglycerol is a physiologic activator of PKC. [21]Thus endothelin-1 may mobilize cytosolic Ca sup 2+ from both the extracellular space and intracellular stores in A10 cells. In the present study, we found that propofol suppresses the endothelin-1-induced Ca²+ influx directly and that it also suppresses the formation of inositol phosphates in these cells. This suppression of endothelin-1-induced formation of inositol phosphates indicates a reduction in the release of Ca²+ from its intracellular stores. Therefore our results suggest that propofol attenuates the endothelin-1-induced mobilization of Ca²+ from both the extracellular space and the intracellular stores in A10 cells.

Research has shown that endothelin-1 activates phosphatidylcholine-hydrolyzing phospholipase D in primary cultured aortic smooth muscle cells and A10 cells, [32,36]and that this activation is independent of PKC activation through phosphoinositide-hydrolyzing phospholipase C. We found that endothelin-1 stimulated choline formation in A10 cells. It is recognized that phospholipase D catalyzes the hydrolysis of phosphatidylcholine, resulting in the formation of phosphatidic acid and choline. [21,23]Phosphatidic acid is subsequently converted to diacylglycerol by the action of phosphatidate phosphohydrolase. [21,23]Thus, it is probable that, in A10 cells, PKC is activated by endothelin-1 via phosphoinositide hydrolysis involving phospholipase C and phosphatidylcholine hydrolysis involving phospholipase D. Thus, in this study, we found that propofol inhibits the endothelin-1-induced formation of inositol phosphates and choline. Based on our findings, it is most likely that, in A10 cells, propofol inhibits endothelin-1-induced activation of PKC by suppressing both phosphoinositide hydrolysis by phospholipase C and phosphatidylcholine hydrolysis by phospholipase D.

Next we showed that propofol had no effect on the NaF-induced formation of inositol phosphates. Endothelin-1 binds to the ETA receptor, which is coupled to phosphoinositide-hydrolyzing phospholipase C by a pertussis toxin-insensitive GTP-binding protein in A10 cells. [31]Heterotrimeric GTP-binding proteins function as intermediaries in transmembrane signaling from receptor to phosphoinositide-hydrolyzing phospholipase C. [33]NaF is a membrane-permeable activator of heterotrimeric GTP-binding proteins. [33]Thus our result makes it unlikely that the effect of propofol is exerted at a point downstream from the GTP-binding protein. However, investigators recently reported that NaF activates not only heterotrimeric GTP-binding protein but also phosphoinositide-hydrolyzing phospholipase C. [44]Propofol likely acts at the point between heterotrimeric GTP-binding protein and phosphoinositide-hydrolyzing phospholipase C. Consequently, we examined the effect of propofol on endothelin-1 binding to its specific receptor. However, propofol had no effect on such endothelin-1 binding. These results strongly suggest that the inhibitory effect of propofol seen in A10 cells is exerted at a point between the ETA receptor and the GTP-binding protein. It is possible that propofol changes the affinity of the GTP-binding protein for the ETA receptor either directly or perhaps indirectly through some unknown compound that is a target for propofol itself.

An intrinsic function of vascular smooth muscle cells is to play an important role in regulating vascular tone. [26]As part of the intracellular signaling system in vascular smooth muscle cells, mobilization of cytosolic Ca²+ and sustained PKC activation have crucial roles in the induction of vasoconstriction. [21,25]In this study, we showed that propofol suppresses the Ca²+ mobilization and PKC activation induced by endothelin-1 in A10 cells. Based on our findings, it is most likely that such a suppression would induce a relaxation of blood vessels preconstricted by endothelin-1 and thus tend to cause a decrease in blood pressure in vivo whenever an endothelin-1-induced vasoconstriction is present.

The peak plasma concentration of propofol has been reported to be 4–10 micro gram/ml (approximately 5–50 micro Meter) in patients in whom general anesthesia was induced with a bolus injection of propofol. [45]Furthermore, it has been estimated that about 97% of propofol is bound to plasma proteins. [45,46]The inhibitory effect of propofol in the present study was observed at concentrations higher than those used clinically. It has been reported that between 5 micro Meter and 7 micro Meter propofol will relax isolated coronary arteries preconstricted with endothelin. [47]These propofol concentrations are also greater than those used clinically. Therefore, this result is consistent with our findings. We speculate that propofol decreases the endothelin-1-induced constriction in the isolated aorta through the inhibitory effects on Ca²+ mobilization and PKC activation observed in the present study.

Endothelin-1 is constitutively released from vascular endothelial cells. [27]In addition, it has been reported that a sustained increase in arterial pressure develops when endothelin-1 is administered by intravenous bolus injection in rats. [28]Plasma endothelin concentrations are reported to be increased during surgery. [29]However, the maximum levels are less than 4 pM. [29]The concentrations of endothelin that we used and in isolated arteries far exceeds in vivo values. In vitro, it has been reported that the significant effect of nifedipine, known to be a Ca²+ blocker, on endothelin-induced Ca²+ influx from the extracellular space is observed at 5 micro Meter in A10 cells. [48]On the other hand, in vivo, the clinically effective concentration of nifedipine in humans is 0.043 micro Meter. [49]Thus we think that the need for high concentrations of endothelin-1 and propofol in vitro is due to its condition compared with in vivo conditions.

In conclusion, these results suggest that propofol inhibits endothelin-1-induced intracellular signaling in vascular smooth muscle cells. The inhibitory effect of propofol that is observed might be exerted at a point between the endothelin-1 receptor and its GTP-binding protein. However, because all of the significant effects are observed at high concentrations, clinical relevance is unclear.