Pentobarbital, but not Propofol, Suppresses Vasopressin-stimulated Heat Shock Protein 27 Induction in Aortic Smooth Muscle Cells

Arginine vasopressin was purchased from Peptide Institute Inc. (Minoh, Japan). Pentobarbital was purchased from Nacalai Tesque, Inc. (Tokyo, Japan). Propofol was purchased from Aldrich (Tokyo, Japan). 12-o -tetradecanoylphorbol 13-acetate (TPA) was purchased from Sigma Chemical Co. (St. Louis, MO). HSP70 antibodies were purchased from Santa Cruz (Santa Cruz, CA). An ECL Western blotting detection system was obtained from Amersham Japan (Tokyo, Japan). Other materials and chemicals were obtained from commercial sources. Vasopressin and pentobarbital were 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% bovine serum albumin (BSA). We used the assay buffer containing 0.01% BSA as the vehicle control for pentobarbital. Propofol was dissolved in ethanol. The maximum concentration of ethanol in the culture medium was 0.1%, and this did not affect the immunoassay of HSP27. We used the assay buffer containing 0.01% BSA and 0.1% ethanol as the vehicle control for propofol.

A10 cells derived from rat aortic smooth muscle cells 28were obtained from the American Type Culture Collection (Rockville, MD). The cells (1 × 10⁵) were seeded into 35- or 90-mm-diameter dishes and maintained at 37°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 later. The cells were treated with pentobarbital or propofol in serum-free Dulbecco’s modified Eagle’s medium for the indicated periods.

Cultured cells were stimulated by vasopressin for 48 h. The stimulated cells were rinsed twice with the assay buffer, lysed, homogenized and sonicated in a lysis buffer containing 62.5 mm Tris/Cl, pH 6.8, 2% sodium dodecyl sulfate, 50 mm dithiothreitol, and 10% glycerol. The cytosolic fraction was collected as the supernatant after centrifugation at 125,000 g  for 10 min at 4°C. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis was performed by the method of Laemmli 29in 10% polyacrylamide gel. Western blotting analysis was performed as described previously 19,20using HSP70 antibodies or HSP27 antibodies, with peroxidase-labeled antibodies raised in goat against rabbit immunoglobulin G being used as second antibodies. Peroxidase activity on the nitrocellulose sheet was visualized on x-ray film by means of the ECL Western blotting detection system.

The concentrations of HSP27 in soluble extracts of the cells were determined by means of a sandwich-type enzyme immunoassay, as described previously. 27Cultured cells were pretreated with pentobarbital (0.1–0.8 mm), propofol (1 μm to 0.1 mm), or vehicle and then stimulated by vasopressin (0.1 μm) or TPA (0.1 μm) for the indicated periods in 1 ml of serum-free Dulbecco’s modified Eagle’s medium. The stimulated cells were washed twice with 1 ml of phosphate-buffered saline and frozen at −80°C for a few days before analysis. The frozen cells on each dish were collected and suspended in 0.3 ml of phosphate-buffered saline, then each suspension was sonicated and centrifuged at 125,000 g  for 20 min at 4°C. The supernatant was used for the immunoassay of HSP27. In brief, we used an enzyme immunoassay system that employs polystyrene balls (3.2 mm in diameter; Immuno Chemicals, Okayama, Japan) carrying immobilized F(ab′)2 fragments of antibody and the same Fab′ fragments labeled with β-d-galactosidase from Escherichia coli . A polystyrene ball carrying antibodies was incubated either with the purified standard for HSP27 or with an aliquot of the samples. This incubation was conducted at 30°C for 5 h in a final volume of 0.5 ml of 10 mm sodium phosphate buffer, pH 7.0, containing 0.3 m NaCl, 0.5% hydrolyzed gelatin, 0.1% BSA, 1 mm MgCl²and 0.1% NaN³. After washing, each ball was incubated at 4°C overnight with 1.5 mU of galactosidase-labeled antibodies in a volume of 0.2 ml with 10 mm sodium phosphate buffer, pH 7.0, containing 0.1 m NaCl, 1 mm MgCl², 0.1% BSA, and 0.1% NaN³. The galactosidase activity bound to the ball was assayed using a fluorogenic substrate, 4-methylumbelliferyl-β-d-galactoside.

Cultured cells were pretreated with pentobarbital (0.8 mm) for 20 min and then stimulated by vasopressin (0.1 μm) in 1 ml of serum-free Dulbecco’s modified Eagle’s medium. After 12 h, the stimulated cells were washed twice with 1 ml of phosphate-buffered saline and frozen at −80°C for a few days before analysis. Total RNA was isolated using a QuickPrep Total RNA Extraction kit (Pharmacia Biotech, Tokyo, Japan). Twenty micrograms of total RNA was then subjected to electrophoresis on a 0.9% agarose/2.2 m formaldehyde gel and blotted onto a nitrocellulose membrane. For Northern blot, the membrane was allowed to hybridize with a cDNA probe that had been labeled using a Multiprime DNA labeling system (Amersham, Buckinghamshire, United Kingdom), as described previously. 30A Bam  HI-Hind  III fragment of cDNA for mouse HSP27 was provided by Dr. L. F. Cooper of the University of North Carolina. 31 

Protein concentrations in soluble extracts were determined using a protein assay kit (Bio-Rad, Hercules, CA), with BSA as the standard protein. Rat HSP27, which was used as the standard for the immunoassay, was purified from skeletal muscle. 19,20 

The data were analyzed by analysis of variance followed by the Student-Newman-Keuls test. P  values < 0.05 were considered significant. All data are presented as the mean ± SD of triplicate determinations.

Vasopressin had little effect on the levels of HSP70 in vascular smooth muscle A10 cells while increasing the levels of HSP27 in the same sample (fig. 1).

Pentobarbital (0.4 mm), which by itself had little effect on the basal levels of HSP27, significantly (time-dependently up to 48 h) inhibited the HSP27 accumulation induced by vasopressin (0.1 μm;fig. 2A). On the contrary, propofol (0.1 mm) did not affect the vasopressin-stimulated accumulation of HSP27 up to 48 h (data not shown). The inhibitory effect of pentobarbital on the HSP27 accumulation was dose-dependent (0.1–0.8 mm), and it was significant at doses of 0.2 mm or more (fig. 2B). In contrast, propofol had little effect on the HSP27 accumulation induced by vasopressin over the range 1 μm to 0.1 mm (fig. 3). Long-term treatment (48 or 96 h) with pentobarbital did not affect the basal levels of HSP27 (table 1). In addition, pentobarbital reduced the vasopressin-stimulated HSP27 accumulation regardless of whether the cells had or had not been pretreated with pentobarbital (table 1).

We previously showed that vasopressin stimulates the expression levels of mRNA for HSP27. 27We next examined the effect of pentobarbital on the levels of mRNA for HSP27 induced by vasopressin. Pentobarbital (0.8 mm), which alone did not affect the basal levels, significantly reduced the vasopressin (0.1 μm)-stimulated levels of mRNA for HSP27 (fig. 4).

We previously reported that TPA alone, an activator of protein kinase C, 32significantly stimulates HSP27 induction in A10 cells and that vasopressin stimulates the induction of HSP27 through protein kinase C activation. 27Pentobarbital markedly inhibited the HSP27 accumulation induced by TPA (0.1 μm;fig. 5). This inhibitory effect of pentobarbital on the HSP27 accumulation was dose-dependent (0.1–0.8 mm), and it was significant at doses of 0.1 mm or more (fig. 5). In contrast, propofol (0.1 mm) had no such effect (284 ± 18 ng/mg protein for vehicle; 900 ± 144 ng/mg protein for 0.1 μm TPA; 312 ± 46 ng/mg protein for 0.1 mm propofol; 961 ± 228 ng/mg protein for 0.1 μm TPA with 0.1 mm propofol, as measured during a stimulation of 48 h).

We previously showed that in an aortic smooth muscle cell line, A10 cells, vasopressin stimulates the induction of HSP27. 27In the present study, we examined the effects of pentobarbital and propofol on the HSP27 induction stimulated by vasopressin in these cells. Pentobarbital significantly reduced the vasopressin-induced accumulation of HSP27 in a time- and dose-dependent fashion. In contrast, propofol had no such effect. In addition, we demonstrated that pentobarbital suppressed the vasopressin-stimulated expression levels of mRNA for HSP27. Thus, our findings suggest that pentobarbital, but not propofol, inhibits the vasopressin-stimulated HSP27 induction in aortic smooth muscle cells.

High-molecular-weight HSPs such as HSP70 are well known to act as molecular chaperones. 14It is recognized that low-molecular-weight HSPs such as HSP27 may also act as chaperones. 14,21It has been shown that heat stress induces both HSP70 and HSP27 in vascular smooth muscle cells. 14In addition, an expression of HSP27 is reportedly induced in aortic smooth muscle cells in restrained rats. 14In our study, vasopressin did not affect the levels of HSP70, but it did stimulate the levels of HSP27 in A10 cells. As for the roles played by HSP27 in the vascular system, it has recently been reported that thrombin and serotonin induce contraction of vascular smooth muscle, and that the contraction is associated with an increased phosphorylation of HSP27. 25,26These findings lead us to speculate that HSP27 may have important effects on aortic smooth muscle cell functions after stimulation by vasoactive agents. It is well recognized that the administration of pentobarbital or propofol reduces systemic blood pressure. 2,3These anesthetics have direct and indirect effects on the cardiovascular system. 2,3As for the direct effect on the cardiovascular system, it has been shown that they reduce the systemic vascular resistance through affecting vascular smooth muscle cells. 2,3,33,34Investigations of HSPs, especially HSP27, in vascular smooth muscle cells suggest that HSP27 may contribute to physiologic and pathologic processes such as contraction, atherosclerosis, and aging of the vasculature. 14In addition, intravenous anesthetics are administered to patients suffering from various stresses and complications. Consequently, further investigations of the relationship between intravenous anesthetics and HSPs in vascular smooth muscle cells could make an important contribution to clarify the exact mechanism underlying the effects of these agents on the vascular system.

In the present study, we showed that pentobarbital also reduced the TPA-induced accumulation of HSP27. We previously reported that TPA alone significantly stimulates HSP27 induction in A10 cells and that protein kinase C activation is involved in the vasopressin-stimulated induction of HSP27. 27Therefore, it is probable that the inhibitory effect of pentobarbital is exerted at a point downstream from protein kinase C in these cells. On the other hand, in the present study, propofol did not affect the accumulation of HSP27 induced by vasopressin or TPA. We have reported that in A10 cells, propofol inhibits protein kinase C activation through a suppression of the activation of both phosphatidylinositol-hydrolyzing phospholipase C and phosphatidylcholine-hydrolyzing phospholipase D by endothelin-1 and vasopressin. 35,36Clinically, propofol decreases systemic blood pressure after a bolus injection. 3However, systemic blood pressure remains stable at the lower level during continuous infusion. 37,38Therefore, it is conceivable that propofol inhibits short-acting signaling by vasoactive agents, resulting in a more-or-less immediate suppression of systemic blood pressure, but does not affect long-acting signaling, such as the induction of HSP27 seen in aortic smooth muscle cells.

Pentobarbital is used to control intracranial pressure, with serum concentrations reported to reach as high as 0.45 mm during a 4-mg · kg⁻¹· h⁻¹continuous infusion. 1It has been shown that the protein binding of pentobarbital is 60%. 39Thus, the free concentration of pentobarbital could reach 0.18 mm. If this is so, it is probable that a suppression of the induction of HSP27 by pentobarbital would occur in vascular smooth muscle cells at the doses used clinically. On the other hand, the plasma concentration of propofol has been reported to be 56–190 μm in patients in whom general anesthesia is maintained with continuous intravenous infusion of propofol alone. 40It has been shown that approximately 97% of propofol is bound to plasma proteins. 41Thus, free concentration of propofol is estimated to be 2–6 μm. In the current study, propofol had no effect on the vasopressin-stimulated HSP27 induction over the range 1 μm to 0.1 mm. Therefore, it is possible that propofol dose not affect on the induction of HSP27 stimulated by vasopressin, not only at doses use clinically, but also at the doses over clinical concentration.

In conclusion, these results suggest that pentobarbital suppresses the vasopressin-stimulated HSP27 induction in vascular smooth muscle cells. This inhibitory effect is probably exerted at a point downstream from protein kinase C.

The authors thank Kouseki Hirade and Hidenori Ito for skillful technical assistance.