Effect of Propofol on Arachidonate Cascade by Vasopressin in Aortic Smooth Muscle Cells : Inhibition of PGI2Synthesis

[5,6,8,9,11,12,14,15-(³) H]AA (208 Ci/mmol), myo-[(³) H]-inositol (81.5 Ci/mmol), [methyl-(³) H]choline chloride (85 Ci/mmol), and a 6-keto PGF¹[small alpha, Greek] radioimmunoassay kit were obtained from Amersham Japan (Tokyo, Japan). Arginine vasopressin was purchased from Peptide Institute (Minoh, Japan). 1-(6((17[small beta, Greek]-3-Methoxyestra-1,3,5,(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U-73122), a phospholipase C inhibitor, [30]was purchased from Funakoshi Pharmaceutical (Tokyo, Japan). D,L-Propranolol hydrochloride (propranolol), a phosphatidic acid phosphohydrolase inhibitor, [31,32]was purchased from Wako Pure Chemical (Osaka, Japan). Propofol was purchased from Aldrich (Tokyo, Japan). Essentially fatty acid-free bovine serum albumin (BSA) and quinacrine, a phospholipase A²inhibitor, [33]were purchased from Sigma Chemical (St. Louis, MO). Other materials and chemicals were obtained from commercial sources.

Vasopressin was dissolved in an assay buffer (consisting of 5 mM HEPES, pH 7.4; 150 mM NaCl; 5 mM KCl; 0.8 mM MgSO⁴; 1 mM CaCl²; and 5.5 mM glucose) containing 0.01% BSA. We used this BSA-containing assay buffer as the vehicle control for vasopressin. U-73122, propranolol, and quinacrine were each dissolved in dimethyl sulfoxide. Propofol was dissolved in ethanol. The maximum concentration of ethanol or dimethyl sulfoxide in the culture medium was 0.1%, and this did not affect the detection of AA release, measurement of PGI²synthesis, or the formation of inositol phosphates and choline. We used assay buffer containing 0.01% BSA and 0.1% dimethyl sulfoxide as the vehicle control for U-73122, propranolol, and quinacrine. We used assay buffer containing 0.01% BSA and 0.1% ethanol as the vehicle control for propofol.

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

As previously described, [25]cultured cells were labeled with [(³) H]AA (0.5 [micro sign]Ci/dish) for 48 h. The labeled cells were washed four times with 1 ml of the assay buffer and subsequently preincubated in 1 ml of assay buffer containing 0.1% essentially fatty acid-free BSA at 37 [degree sign]C for 20 min. The cells were then stimulated by various doses of vasopressin. After 30 min, the medium was collected and its radioactivity was determined. To determine whether activation of phosphoinositide-hydrolyzing phospholipase C, phosphatidylcholine-hydrolyzing phospholipase D, or phospholipase A²are involved in vasopressin-induced AA release and PGI (²) synthesis in A10 cells, we evaluated the effects of U-73122, propranolol, or quinacrine, respectively, on vasopressin-induced AA release and PGI²synthesis. Pretreatment with U-73122 (0.3-10 [micro sign]M), propranolol (5-300 [micro sign]M), quinacrine (5-30 [micro sign]M), or vehicle was performed for 20 min.

As previously described, [25]PGI²synthesis was determined using methods similar to those described in the section “assay for AA release,” except that unlabeled cells were used. Cultured cells were pretreated with U-73122 (0.3-10 [micro sign]M), propranolol (5-300 [micro sign]M), quinacrine (5-30 [micro sign]M), propofol (0.1 [micro sign]M-0.1 mM), or vehicle for 20 min and then stimulated by various doses of vasopressin. After the indicated period, the medium was collected, and the level of 6-keto PGF¹[small alpha, Greek](a stable and inactive metabolite of PGI²)[27,34]in the medium was determined by means of an radioimmunoassay kit. Briefly, an assay buffer (0.05 M phosphate buffer, pH 7.3, containing 0.05% BSA and 0.1% sodium azide), the medium, the 6-keto PGF¹[small alpha, Greek] tracer (6-keto PGF¹[small alpha, Greek][(¹²⁵) I]iodotyrosine methyl ester in ethanol and water) and the 6-keto PGF¹[small alpha, Greek] antiserum were transferred to tubes to produce a final ratio 2:1:1:1. The contents were mixed and then incubated over 15 h at 4 [degree sign]C. Amerlex [trade mark sign]-M second antibody reagent was added to the tubes. The tubes were centrifuged at 14,000g for 10 min, and the supernatants were discarded. Then, the radioactivity was determined. The value of the samples were calculated from the standard curve. The cross-reactivity between 6-keto PGF¹[small alpha, Greek] and other prostaglandins was less than 1%.

As previously described, [35,36]cultured cells were labeled with myo-[(³) H]inositol (3 [micro sign]Ci/dish) for 48 h. The labeled cells were preincubated with 20 mM LiCl for 10 min at 37 [degree sign]C in 1 ml of assay buffer containing 0.01% BSA. The cells were stimulated by vasopressin for 20 min after pretreatment of U-73122 or 150 min after pretreatment of propofol. The reaction was discontinued by adding 1 ml trichloroacetic acid, 30%. The acidic supernatant was treated with diethyl ether to remove the acid and then neutralized with 0.1 N NaOH. The supernatant was applied to an anion exchange column containing 1 ml of Dowex AG1-X8 (100-200 mesh, formate form; Bio-Rad Laboratories, Hercules, CA). The radioactive inositol phosphates were eluted with 8 ml formic acid, 0.1 M, containing 1 M ammonium formate. [35,36]Pretreatment with propofol (0.1 [micro sign]M-0.1 mM), U-73122 (0.3-10 [micro sign]M), or vehicle was performed for 20 min.

As previously described, [37]to help determine the phosphatidylcholine-hydrolyzing phospholipase D activity in A10 cells, cultured cells were labeled with [methyl-(³) H]choline chloride (3 [micro sign]Ci/dish) for 48 h. The labeled cells were washed twice with 1 ml of the assay buffer and the pretreated with propofol (0.1 [micro sign]M-0.1 mM) or vehicle for 20 min at 37 [degree sign]C in 1 ml of assay buffer containing 0.01% BSA. They were then stimulated by vasopressin for 150 min until the reaction was discontinued by adding 0.75 ml of ice-cold methanol. The dishes were then placed on ice for 30 min, and the contents were transferred subsequently to tubes to which chloroform was added. They were then left standing on ice for 60 min, whereupon chloroform and water were added to produce 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 of Dowex 50-WH⁺(Bio-Rad Laboratories, 200-400 mesh, Hercules, CA) as described, [37]with a minor modification. [15]Briefly, the phase was diluted to 5 ml with water and applied to the column. Glycerophosphocholine and choline phosphate were removed with 24 ml of water, and radioactive choline was then eluted with 10 ml HCl, 1 M.

The radioactivity of³H 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 by 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.

It has been reported that vasopressin stimulates AA release in a time- and dose-dependent manner in A10 cells. [38]We confirmed that vasopressin significantly increased AA release in a dose-dependent manner over the range 0.1 nM to 0.1 [micro sign]M, the maximum effect being observed at 0.1 [micro sign]M (Figure 1).

It has also been reported that vasopressin stimulates 6-keto PGF (¹)[small alpha, Greek] synthesis in a time-dependent manner in A10 cells. [39]Vasopressin (0.1 [micro sign]M) increased 6-keto PGF¹[small alpha, Greek] synthesis in a time-dependent manner (up to 180 min) in A10 cells (Figure 2A). Significant 6-keto PGF¹[small alpha, Greek] production occurred when vasopressin-stimulation was continued for 120 min or more. The stimulatory effect of vasopressin on 6-keto PGF¹[small alpha, Greek] synthesis was dose-dependent over the range 0.1 nM to 0.1 [micro sign]M (Figure 2B), the maximum effect being observed at 10 nM.

It has been reported that vasopressin induces phospholipase C-catalyzed phosphoinositide hydrolysis in A10 cells [20,40]and that phosphoinositide hydrolysis results in the formation of diacylglycerol and inositol phosphates. [21,40]First, we confirmed that vasopressin induces the formation of inositol phosphates in these cells (Figure 3). Vasopressin induced the formation of inositol phosphates in a time dependent manner (up to 150 min). U-73122 is known to be a potent inhibitor of phosphoinositide-hydrolyzing phospholipase C. [30]To establish whether activation of phosphoinositide-hydrolyzing phospholipase C is indeed involved in the stimulation of AA release by vasopressin in A10 cells, we evaluated the effect of U-73122 on vasopressin-induced AA release. Pretreatment with 3 [micro sign]M U-73122, which by itself had little or no effect on AA release, significantly inhibited vasopressin (0.1 [micro sign]M)-induced AA release (Table 1).

We also found that pretreatment with U-73122, which by itself had little effect on the formation of inositol phosphates, significantly inhibited the vasopressin (0.1 [micro sign]M)-induced formation of inositol phosphates in A10 cells. This inhibitory effect of U-73122 (10 [micro sign]M) was significant and dose-dependent over the range 1 to 10 [micro sign]M, the maximum effect being observed at 10 [micro sign]M (Table 2).

U-73122, at a dose that by itself had little effect on 6-keto PGF1 [small alpha, Greek] synthesis, significantly reduced vasopressin

U-73122, at a dose that by itself had little effect on 6-keto PGF1[small alpha, Greek] synthesis, significantly reduced vasopressin (0.1 [micro sign]M)-induced 6-keto PGF1[small alpha, Greek] synthesis (10.0 +/− 0.27 ng/dish for 0.1 [micro sign]M vasopressin with vehicle pretreatment; 5.7 +/− 0.49# ng/dish for 0.1 [micro sign]M vasopressin with 1 [micro sign]M U-73122 pretreatment, values for vasopressin-unstimulated cells were subtracted to produce each data point).

It has been reported that vasopressin induces phospholipase D-catalyzed phosphatidylcholine hydrolysis in A10 cells [23]and that phosphatidylcholine hydrolysis by phospholipase D results in the formation of phosphatidic acid and choline. [41-43]First, we confirmed that vasopressin induces the formation of choline in these cells (23,503 +/− 1,245 cpm for vehicle; 35,140 +/− 1,087** cpm for 0.1 [micro sign]M vasopressin). Vasopressin (0.1 [micro sign]M) induced the formation of choline in a time-dependent manner (up to 150 min). To clarify whether activation of phosphatidylcholine-hydrolyzing phospholipase D is involved in the stimulation of AA release and PGI²synthesis by vasopressin in A10 cells, we evaluated the effect of propranolol (an inhibitor of phosphatidic acid phosphohydrolase, [31,32]which converts phosphatidic acid to diacylglycerol [41-43]) on vasopressin-induced AA release and 6-keto PGF (¹)[small alpha, Greek] synthesis. Pretreatment with 300 [micro sign]M propranolol, which by itself had little or no effect on AA release, significantly inhibited the vasopressin (0.1 [micro sign]M)-induced AA release (Table 3). In addition, propranolol, at doses that by themselves had no detectable effect on 6-keto PGF¹[small alpha, Greek] synthesis, significantly reduced vasopressin (0.1 [micro sign]M)-induced 6-keto PGF¹[small alpha, Greek] synthesis in a dose-dependent manner (Figure 4).

We reported previously that propofol inhibits the formation of inositol phosphates that is induced by endothelin-1 in A10 cells. [15]In the current study, pretreatment with propofol, which by itself had no detectable effect on inositol phosphates formation, inhibited the vasopressin (0.1 [micro sign]M)-induced formation of inositol phosphates. The only significant effect of propofol on inositol phosphates formation was seen at a dose of 0.1 mM (a 40% reduction in the effect of vasopressin, Figure 3).

Pretreatment with propofol, which by itself had little effect on the formation of choline, significantly inhibited the vasopressin (0.1 [micro sign]M)-induced formation of choline. At 0.1 mM, propofol reduced the vasopressin (0.1 [micro sign]M)-induced choline formation by approximately 40%(11,560 +/− 2,671 cpm for 0.1 [micro sign]M vasopressin with vehicle pretreatment; 4,480 +/− 2,898[dagger][dagger] cpm for 0.1 [micro sign]M vasopressin with 0.1 mM propofol pretreatment, values for vasopressin-unstimulated cells were subtracted to produce each data point).

Pretreatment with propofol (10 [micro sign]M), which by itself had no detectable effect on 6-keto PGF¹[small alpha, Greek] synthesis, significantly attenuated the time-dependent (up to 180 min) stimulation by vasopressin (0.1 [micro sign]M) of 6-keto PGF¹[small alpha, Greek] synthesis (Figure 2A). The effect of propofol on 6-keto PGF¹[small alpha, Greek] synthesis was significant at doses more than 10 [micro sign]M (Figure 5).

It has been reported that phospholipase A²activation is involved in vasopressin-induced AA release in A10 cells. [28]We confirmed that quinacrine, known to be a phospholipase A²inhibitor, [33]at a dose that by itself had little effect on AA release or 6-keto PGF¹[small alpha, Greek] synthesis, significantly inhibited the vasopressin-induced AA release (data not shown) and 6-keto PGF¹[small alpha, Greek] synthesis (10.7 +/− 0.37 ng/dish for 0.1 [micro sign]M vasopressin with vehicle pretreatment; 6.3 +/− 1.1 [double dagger][double dagger] ng/dish for 0.1 [micro sign]M vasopressin with 30 [micro sign]M quinacrine pretreatment, values for vasopressin-unstimulated cells were subtracted to produce each data point).

In the current study, we demonstrated that vasopressin-induced AA release was dose-dependently reduced by U-73122, an inhibitor of phosphoinositide-hydrolyzing phospholipase C, [30]in a rat aortic smooth muscle cell line: A10 cells. It has been reported that vasopressin induces phosphoinositide-hydrolysis, resulting in the formation of diacylglycerol and inositol phosphates. [21,40]Diacylglycerol and inositol 1,4,5-trisphosphate then serve as messengers for the activation of protein kinase C and the mobilization of intracellular Ca²⁺, respectively. [20,21,40]In addition, diacylglycerol, which is a source of AA, is converted to monoacylglycerol and fatty acids in A10 cells. [44]Thus, our findings suggest that phosphoinositide hydrolysis by phospholipase C is involved in the stimulation of AA release by vasopressin in these cells. We next showed that propranolol, an inhibitor of phosphatidic acid phosphohydrolase, [31,32]also significantly inhibited vasopressin-induced AA release in A10 cells. It has been reported that vasopressin stimulates phosphatidylcholine-hydrolyzing phospholipase D in A10 cells, [22,23]in which phosphatidylcholine breakdown is downstream from phosphoinositide-hydrolysis by phospholipase C. [23]Phosphatidylcholine is hydrolyzed by phospholipase D, resulting in the formation of choline and phosphatidic acid, which is further degraded to diacylglycerol by phosphatidic acid phosphohydrolase. [41-43]Therefore, to judge from our results, the conversion of phosphatidic acid to diacylglycerol is involved in vasopressin-induced AA release in A10 cells. It has been reported that phospholipase A²plays an important role in vasopressin-induced AA release in A10 cells. [28]We confirmed that quinacrine, a phospholipase A (²) inhibitor, [33]suppressed vasopressin-induced AA release in these cells. On the basis of our current findings, we suggest that phosphoinositide-phospholipase C and phosphatidylcholine-phospholipase D (and phospholipase A²) are involved in vasopressin-induced AA release in A10 cells.

PGI²is well known to be a major eicosanoid product derived from AA in vascular smooth muscle cells. [27]In the current study, we demonstrated that vasopressin stimulates PGI²synthesis in A10 cells and that U-73122 and propranolol both significantly reduced this vasopressin-induced PGI²synthesis, and AA release, in these cells. In addition, we confirmed that quinacrine inhibited vasopressin-induced PGI²synthesis in A10 cells. Our findings, therefore, strongly suggest that phosphoinositide hydrolysis by phospholipase C and phosphatidylcholine hydrolysis by phospholipase D are involved in the mechanism by which vasopressin induces PGI²synthesis in A10 cells.

In the current study, we showed that propofol suppressed vasopressin-induced PGI²synthesis in A10 cells. In these cells, it has been reported that the activation of phosphatidylcholine-hydrolyzing phospholipase D by vasopressin is kinetically downstream from the initial phosphoinositide hydrolysis by phospholipase C, and that it involves the intermediate activation of protein kinase C. [23]We demonstrated that propofol suppressed the vasopressin-induced formation of inositol phosphates and choline in these cells. We reported previously [15]that propofol suppresses the endothelin-1-induced formation of inositol phosphates and choline formation in A10 cells. Our current findings suggest that propofol suppresses the stimulation by vasopressin of the arachidonate cascade, at least partly, by inhibiting phosphoinositide-hydrolyzing phospholipase C and phosphatidylcholine-hydrolyzing phospholipase D in A10 cells. However, the inhibition by propofol of the activation of phosphoinositide-hydrolyzing phospholipase C or phosphatidylcholine-hydrolyzing phospholipase D was partial. In contrast, we showed that 0.1 [micro sign]M propofol suppressed vasopressin-induced PGI²synthesis almost completely. Therefore, it is likely that an inhibition of phospholipase A²is also involved in the suppression of PGI²synthesis by propofol. The effects of propofol on intracellular signaling system in vascular smooth muscle cells shown in our current and in previous studies [15]are shown in Figure 6.

The vascular action of propofol has been reported to be caused by its direct effects on endothelial cells and vascular smooth muscle cells [10-14]and by indirect effects such as those exerted via the central nervous system. [7]We have shown, using the A10 cell line, that propofol suppresses the intracellular signaling responsible for the contraction of vascular smooth muscle. Because the current results clearly indicate that propofol also inhibits vasopressin-induced PGI²synthesis, it can be assumed that propofol will modulate the effects of vasodilators and of vasoconstrictors. The vasoconstrictive effect of vasopressin may be mediated by diacylglycerol and inositol trisphosphate produced by phospholipases C and D, and this vasoconstrictive effect of vasopressin is self-modulated by subsequent release of PGI². The vasorelaxing effect of propofol may in part be explained by its inhibition of vasopressin-mediated activation of phospholipase C and phospholipase D. Such relaxation may partially be offset by the simultaneous effect of propofol to attenuate vasopressin-stimulated PGI²synthesis. Mobilization of cytosolic Ca²⁺and sustained protein kinase C activation induced by phosphoinositide-phospholipase C and phosphatidylcholine-phospholipase D have crucial roles in the induction of vasoconstriction. [17,21]However, AA release and 6-keto PGF¹[small alpha, Greek] synthesis means PGI²synthesis in cultured aortic smooth muscle cells does not occur immediately after stimulation. [38,39]In the current study, significant 6-keto PGF¹[small alpha, Greek] production began at 120 min after vasopressin-stimulation in A10 cells. Clinically, propofol is used not only for induction of anesthesia, but also for maintenance by continuous intravenous administration during several hours. [1]In the current study, propofol-treatment was continued for 170 min. Therefore, it is possible that the suppression of vasopressin-induced PGI²synthesis by propofol has a role in automodulating the propofol-induced vasodilation during long-time anesthesia rather than during bolus infusion.

The plasma concentration of propofol recently has been reported to be 56-190 [micro sign]M in patients in whom general anesthesia was maintained with continuous intravenous infusion of propofol alone. [45]In addition, it has been shown that approximately 97% of propofol is bound to plasma proteins. [46]Therefore, free concentration of propofol is estimated to be 2-6 [micro sign]M. The inhibitory effect of propofol in the current study was observed at concentrations higher than those used clinically. However, the concentration of vasopressin that we used far exceeds in vivo values. Therefore, it is possible that the need for high concentrations of vasopressin and propofol in vitro may be because of its condition compared with in vivo conditions.

In conclusion, these results suggest that propofol suppresses the stimulation of the arachidonate cascade by vasopressin at least partly by inhibiting phosphoinositide-phospholipase C and phosphatidylcholine-phospholipase D. Propofol-mediated inhibition of vasopressin-stimulated synthesis of PGI²may reduce the vasorelaxation by propofol.

# P < 0.05 versus the value for vasopressin without U-73122 pretreatment.

** P < 0.05 versus the value for vehicle.

[dagger][dagger] P < 0.05 versus the value for vasopressin without propofol pretreatment.

[double dagger][double dagger] P < 0.05 versus the value for vasopressin without quinacrine pretreatment.