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Meeting Abstracts  |   June 1998
Effect of Propofol on Norepinephrine-induced Increases in [Ca2+](i) and Force in Smooth Muscle of the Rabbit Mesenteric Resistance Artery 
Author Notes
  • (Imura) Graduate Student, Department of Anesthesiology and Resuscitology, Nagoya City University Medical School.
  • (Shiraishi) Graduate Student, Department of Anesthesiology and Critical Care Medicine, Kagoshima University Medical School.
  • (Katsuya) Chairman and Professor, Department of Anesthesiology and Resuscitology, Nagoya City University Medical School.
  • (Itoh) Chairman and Professor, Department of Pharmacology, Nagoya City University Medical School.
Article Information
Meeting Abstracts   |   June 1998
Effect of Propofol on Norepinephrine-induced Increases in [Ca2+](i) and Force in Smooth Muscle of the Rabbit Mesenteric Resistance Artery 
Anesthesiology 6 1998, Vol.88, 1566-1578. doi:
Anesthesiology 6 1998, Vol.88, 1566-1578. doi:
PROPOFOL (2,6-diisopropylphenol) is a widely used intravenous anesthetic agent with a rapid onset, short duration of action, and rapid elimination. [1] Induction of anesthesia with propofol is often accompanied by a mild to more severe hypotension [1,2] caused by decreases in both cardiac output and peripheral vascular resistance. [2–8] Direct evidence of propofol-induced vascular relaxation has been obtained using isolated blood vessels. [9–13] To date, much of the experimental work has focused on the conduit arteries. However, some properties of small arteries, such as membrane properties and agonist-activated Ca2+mobilization mechanisms, differ from those of conduit arteries. [14] As yet, the actions of propofol have not been studied using small splanchnic resistance arteries, which are important in the control of total peripheral resistance and maintenance of splanchnic blood flow in patients having surgery. [15,16] 
The contraction - relaxation cycle in vascular smooth muscle depends largely on the intracellular concentration of Ca2+([Ca2+](i)). An increase in [Ca2+]iinduced by Ca2+-mobilizingagonists in smooth muscle cells can occur through an activation not only of Ca2+influx but of Ca2+release from the intracellular stores. [14] In addition, in contrast to the effect of K+-depolarizations, the effects induced by such agonists include an enhancement of actin-myosin interaction at a given [Ca2+]i(through an agonist-induced increase in myofilament Ca2+sensitivity), which results in a maintained contraction at relatively low [Ca2+]i. [14] Propofol inhibits both high K+-inducedand phenylephrine-induced contractions in the rat thoracic aorta, and the former response is more sensitive to propofol than the latter. [10] Chang and Davis [10] also found that propofol attenuates the Ca2+-inducedcontraction in Ca2+-freesolution containing high K+and suggested that propofol relaxes aortic smooth muscle by blocking L-type Ca2+channels. In contrast, the relaxation induced by propofol in the porcine coronary artery is more profound in preparations contracted by various agonists than in those contracted by high K+, [12] suggesting that an important part of the effect of propofol on Ca2+-influxis exerted through receptor-operated mechanisms. It is a yet unknown which mechanism might be responsible for propofol-induced vascular relaxation in small splanchnic resistance arteries.
In smooth muscle of the rabbit mesenteric resistance artery, norepinephrine induces a two-phase increase in [Ca2+]iand force: The transient phasic response is provoked by Ca2+release from intracellular storage sites, whereas the subsequently maintained tonic phase is induced by an activation of Ca2+influx through the plasmalemma. [17–21] In smooth muscle tissues, ryanodine causes a loss of function of the norepinephrine-sensitive Ca2+storage sites; thus in ryanodine-treated smooth muscle, norepinephrine increases [Ca2+]iand force only through an activation of agonist-activated Ca2+-influx. [18–22] These results suggested to us that the ryanodine-treated smooth muscle strip would be a useful preparation to study the action of propofol on norepinephrine-induced Ca2+influx.
In this study we wanted to clarify the possible mechanisms underlying the propofol-induced vasodilation in the rabbit mesenteric resistance artery. To this end, we first observed the effect of propofol on the contractions induced by norepinephrine (to activate receptor-mediated mechanisms) and high K+(to activate voltage-operated Ca2+channels) in endothelium-denuded strips. The effect of propofol on norepinephrine-induced Ca2+release was examined by observing its action on norepinephrine-induced contractions in Ca2+-freesolution. The effect of propofol on the Ca2+influx activated by high K+or norepinephrine was finally observed by simultaneous measurement of [Ca2+]iand force in ryanodine-treated smooth muscle strips, and the effects of propofol were compared with those of nicardipine.
Materials and Methods
Twenty-six male Japan White albino rabbits (supplied by Kitayama Labes Co., Ina, Japan) that weighed 1.9 to 2.5 kg were anesthetized by injection of pentobarbitone sodium (40 mg/kg given intravenously) and then killed by exsanguination. The protocols used conformed with guidelines on the conduct of animal experiments issued by Nagoya City University Medical School and by the Japanese government (law no. 105, notification no. 6) and were approved by the Committee on the Ethics of Animal Experiments of Nagoya City University Medical School. The third and fourth branches of the mesenteric artery distributing to the region of the ileum (diameter of approximately 70 - 100 [micro sign]m) were excised immediately and cleaned by removal of connective tissue in Krebs solution under a binocular microscope at room temperature. After the artery had been cut open along its long axis using a small scissors, the endothelium was carefully removed by gentle rubbing of the internal surface of the vessel using small pieces of razor blade, as described previously. [18,19] Satisfactory ablation of the endothelium was verified pharmacologically by the absence sence of a relaxing effect of 3 - 10 [micro sign]M acetylcholine during a norepinephrine-induced contraction.
Recording of Mechanical Activity
Fine circularly cut strips (0.2 to 0.3 mm long, 0.04 to 0.05 mm wide, 0.02 to 0.03 mm thick) were prepared for force recording. A fine silk thread was tied to each end of the strip and then fixed to a small piece (about 1 mm x 1 mm) of Scotch double-sided adhesive tape (3M, St. Paul, MN). One tape was fixed to the chamber and the other to the strain gauge (UL-2, Minebea, Tokyo, Japan) to record isometric contraction, as described previously. [17–19] The chamber volume was 0.9 ml, and the solutions were injected rapidly using a syringe from one end of the chamber and simultaneously aspirated by a pump from the other end. The resting force (0.016 +/- 0.005 mN) was adjusted to obtain a maximum contraction in Krebs solution containing 128 mM K+.
The strips were contracted by various concentrations of high-K+or norepinephrine for 2 min at 5-min intervals, using increasing concentration steps, to obtain reproducible responses. The concentration-dependent effects of high-K+and norepinephrine were repeatedly observed in strips that had been pretreated for 5 min with various concentrations of propofol (10 - 100 [micro sign]M), which remained in place throughout the sequence of stimuli.
To observe the effect of propofol on the norepinephrine-induced release of Ca2+from the storage sites, experiments were carried out in Ca2+-freesolution containing 2 mM EGTA. To this end, after a 4-min removal of Ca2+by an application of this Ca2+-freesolution, norepinephrine (3 [micro sign]M) was applied twice with a 2-min interval in the same Ca2+-freesolution. The “first norepinephrine” was applied for 0.5 min after a 4-min removal of Ca2+and then washed out for 2 min using Ca2+-freesolution. Subsequently, the “second norepinephrine” was applied for 2 min, followed by a 1-min washout with Ca2+-freesolution. The strip was brought back to Krebs solution (containing 2.6 mM Ca2+) for 20 min. Propofol (30 or 100 [micro sign]M) was applied in Ca2+-freesolution for 3 min before and was present during the first norepinephrine, and then propofol and norepinephrine were both washed out using Ca2+-freesolution. Two minutes later, the second norepinephrine was applied in Ca2+-freesolution with no further addition of propofol (Figure 1).
Figure 1. Experimental protocol used to investigate the action of propofol on norepinephrine-induced contraction in Ca2+-freesolution (see Materials and Methods for further details).
Figure 1. Experimental protocol used to investigate the action of propofol on norepinephrine-induced contraction in Ca2+-freesolution (see Materials and Methods for further details).
Figure 1. Experimental protocol used to investigate the action of propofol on norepinephrine-induced contraction in Ca2+-freesolution (see Materials and Methods for further details).
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The effect of propofol on caffeine-induced Ca2+release from the storage sites was examined by observing its effect on caffeine-induced contraction in Ca2+-freesolution containing 2 mM EGTA. To this end, after a 4-min application of Ca2+-freesolution, caffeine (10 mM) was applied for 2 min in Ca2+-freesolution, followed by a 1-min washout with Ca2+-freesolution. The strip was brought back to Krebs solution for 20 min. Propofol (100 [micro sign]M) was applied in Ca2+-freesolution for 3 min before and was present during the application of caffeine.
Simultaneous Measurement of [Ca2+]iand Force
[Ca2+]iand isometric force were measured simultaneously using strips prepared as described before. [18–20] For this purpose, the strip was transferred to a chamber with a 0.3-ml volume and mounted horizontally on an inverted microscope (Diaphoto TMD with special optics for epifluorescence; Nikon, Tokyo, Japan). The resting force was adjusted to obtain a maximum contraction in Krebs solution containing 128 mM K+.
To enable loading of Fura 2 into smooth muscle cells of the strip, 1.2 [micro sign]M acetoxymethyl ester of Fura 2 (Fura 2-AM) was applied for 1.5 h in Krebs solution at room temperature. After this period, the solution containing Fura 2-AM was washed out with Krebs solution for 1 h to ensure sufficient esterification of Fura 2-AM in the cells. The position of the strip was adjusted to the center of the field, with a mask placed in an intermediate image plane to reduce background fluorescence (a 0.04-mm square shape). The Fura 2 fluorescence emission at 510 nm (obtained using an interference filter centered at 510 nm and a full width at half transmission of 20 nm) was passed through the lens (X20 CF Fluor objective lens, Nikon) and collected in a photomultiplier tube (R 928, side-on type; Hamamatsu Photonics, Hamamatsu, Japan) via a dichroic mirror (DM-400, Nikon) that was substituted for the photochanger in a Nikon Diaphoto-TMD microscope. Two alternative excitation wavelengths, 340 nm and 380 nm (each slit 5 nm), were applied by a spectrofluorimeter (CAM 220, Japan Spectroscopic Co. Ltd., Tokyo, Japan), and the data were analyzed using software developed in our laboratory. The ratio of the Fura 2 fluorescence intensities excited by 340 or 380 nm was calculated after subtraction of the background fluorescence (<15% of signals), and the [Ca2+iwas calculated according to the formula described by Grynkiewicz et al. [23] and an in vitro calibration procedure. [18,19,24] The ratio of maximum (Fmax) to minimum (Fmin) fluorescence was determined in the calibration solution after subtraction of background, and the 380-nm signal of Fura 2 was assumed to decrease by 15% in the cell because of the possible intracellular viscosity effects of the dye. [18,19,24] The kd for Fura 2 was estimated to be 200 nM. [18,19] This method of calculating [Ca2+]imay not be accurate if Fura 2 binds to some proteins in the cell. [25] To minimize the leakage of Fura 2 from the cells, the present experiment were conducted at room temperature (25 [degree sign]C), and ultraviolet light was applied during the recording period only (to minimize the photobleaching of Fura 2). [18–21] 
To study the effect of propofol on the Ca2+influx induced by norepinephrine or high K+, its effects were observed in ryanodine-treated smooth muscle strips, in which the norepinephrine-sensitive and caffeine-sensitive Ca2+storage sites in the smooth muscle cells are functionally removed. [18,19,26] After the control responses induced by 128 mM K+and 3 [micro sign]M norepinephrine had been recorded, 10 [micro sign]M ryanodine and 10 mM caffeine were applied for 5 min in Krebs solution, followed by a 10-min application of Krebs solution containing 10 [micro sign]M ryanodine alone. High K+(128 mM) and norepinephrine (3 [micro sign]M) were applied for 2-min periods (with a 20-min interval) in the presence of 10 [micro sign]M ryanodine until reproducible responses were obtained. These procedures were repeated in the presence of propofol. Propofol (10 - 100 [micro sign]M) was applied for 5 min before and was present during the application of high K+or norepinephrine.
To study the involvement of L-type Ca2+channels, the propofol-induced inhibition of the norepinephrine-induced increase in [Ca2+]iwas observed in both the presence and absence of nicardipine (an L-type Ca2+channel inhibitor) in ryanodine-treated strips. High K+(128 mM) and norepinephrine (3 [micro sign]M) were successively applied for 2-min periods with a 10-min interval. This protocol was repeated after a 20-min intermission. After the control responses induced by 128 mM K+and 3 [micro sign]M norepinephrine had been recorded, nicardipine (0.3 [micro sign]M) was pretreated for 5 min and norepinephrine and high K+were again applied for 2-min periods in the presence of nicardipine, followed by a 20-min washout with Krebs solution containing nicardipine. Propofol (100 [micro sign]M) was then applied and was present before and during the application of high K+or norepinephrine (in the presence of 0.3 [micro sign]M nicardipine).
Solutions
The Krebs solution was composed of 137.4 mM Na+, 5.9 mM K+, 1.2 mM Mg2+, 2.6 mM Ca2+, 15.5 mM HCO3-, 1.2 mM H2PO4-, 134 mM Cl-, and 11.5 mM glucose. The concentration of K+was modified by the isotonic replacement of NaCl with KCl. Ca2+-freeKrebs solution was made by substituting an equimolar concentration of MgCl2for CaCl2and then adding 2 mM EGTA. All the solutions used in the present experiments contained guanethidine (5 [micro sign]M) and propranolol (3 [micro sign]M) to prevent norepinephrine outflow from sympathetic nerves and [Greek small letter beta]-adrenoceptor stimulation by exogenously applied norepinephrine, respectively. The solutions were bubbled with 95% oxygen and 5% carbon dioxide, and their pH was maintained at 7.3 or 7.4.
The calibration solution for Ca2+measurement contained 11 mM EGTA, 110 mM KCl, 1 mM MgCl2, 20 [micro sign]M Fura 2, and 20 mM HEPES, pH 7.1, with or without 11 mM CaCl2.
Drugs
Drugs used were Fura 2, Fura 2-AM, EGTA, and HEPES (Dojin, Kumamoto, Japan), caffeine (Wako Pure Chemical, Tokyo, Japan), norepinephrine and nicardipine (Sigma Chemical Co., St. Louis, MO), Ryanodine (Agri-system, Wind Gap, PA), guanethidine (Tokyo Kasei, Tokyo, Japan), acetylcholine hydrochloride (Daiichi Pharmaceutical Co., Tokyo, Japan), and propranolol (Nacalai, Kyoto, Japan). Pure propofol (99.9%) was provided by Zeneca Pharmaceuticals (Mereside, Macclesfield, UK). Propofol was dissolved in dimethyl sulfoxide to make a 0.55-M stock solution, which was diluted as required in the Krebs solution. The vehicle (0.02% dimethyl sulfoxide) itself had no effect on the increase in [Ca2+]iand force induced by high K+or norepinephrine.
Statistical Analysis
In the present experiments, most of the results were obtained in both the absence (control) and presence of propofol in one and the same strip unless otherwise noted, with n indicating the number of strips used. The values recorded are expressed as means +/- SD. Statistical analysis was performed using a one-way repeated-measures analysis of variance (Stat View 4.02; Abacus Concepts, Berkeley, CA) followed by the Scheffe F test for post hoc analysis (Super ANOVA, Abacus Concepts), as well as paired or unpaired Student's t tests. Probability values <0.05 were considered significant. The ED50values (the dose that produces 50% of the maximal effect) for the action of propofol on the responses to 128 mM K+and 3 [micro sign]M norepinephrine were obtained by fitting the data points for each strip by a nonlinear least-squares method using software (Kaleida Graph, Synergy Software, Reading, PA) for a Macintosh personal computer (Apple Computer Co., Tokyo, Japan).
Results
Effect of Propofol on Contractions Induced by High K+and Norepinephrine
High K+produced a large phasic followed by a small tonic contraction (Figure 2A) in a concentration-dependent manner. Propofol had no effect on the resting force, but it attenuated both phasic and tonic contraction induced by high K+(Figure 2A, Figure 2B) in a concentration-dependent manner. Norepinephrine produced a phasic and subsequently generated a large tonic contraction in a concentration-dependent manner (Figure 3A). Propofol attenuated both the phasic and tonic contractions induced by norepinephrine (Figure 3), which was also concentration dependent. The ED50value for the effect of propofol on the maximum (phasic) contraction induced by 128 mM K+was 24.3 +/- 0.4 [micro sign]M, and this value was significantly less than that obtained for its effect on the maximum contraction induced by 3 [micro sign]M norepinephrine (36.6 +/- 10.3 [micro sign]M; n = 6, P < 0.03).
Figure 2. Concentration-dependent effect of propofol on contractions induced by various concentrations of high K+. (A) Actual traces of contractions induced by various concentrations of high K+in the absence (left panel) or presence (right panel) of 30 [micro sign]M propofol (both traces were obtained from a single smooth muscle strip). (B) Concentration dependence of the effect of propofol on the maximum (phasic) contractions induced by various concentrations of high K+. The maximum amplitude of contraction induced by 80 mM K+in the absence of propofol was normalized as a relative force of 1.0 for each strip. [black circle], control;[white triangle], 10 [micro sign]M propofol;[white square], 30 [micro sign]M propofol;[black triangle down], 100 [micro sign]M propofol. Mean of data from six strips, with SD shown by vertical lines. *Significant difference from the corresponding control (P < 0.05).
Figure 2. Concentration-dependent effect of propofol on contractions induced by various concentrations of high K+. (A) Actual traces of contractions induced by various concentrations of high K+in the absence (left panel) or presence (right panel) of 30 [micro sign]M propofol (both traces were obtained from a single smooth muscle strip). (B) Concentration dependence of the effect of propofol on the maximum (phasic) contractions induced by various concentrations of high K+. The maximum amplitude of contraction induced by 80 mM K+in the absence of propofol was normalized as a relative force of 1.0 for each strip. [black circle], control;[white triangle], 10 [micro sign]M propofol;[white square], 30 [micro sign]M propofol;[black triangle down], 100 [micro sign]M propofol. Mean of data from six strips, with SD shown by vertical lines. *Significant difference from the corresponding control (P < 0.05).
Figure 2. Concentration-dependent effect of propofol on contractions induced by various concentrations of high K+. (A) Actual traces of contractions induced by various concentrations of high K+in the absence (left panel) or presence (right panel) of 30 [micro sign]M propofol (both traces were obtained from a single smooth muscle strip). (B) Concentration dependence of the effect of propofol on the maximum (phasic) contractions induced by various concentrations of high K+. The maximum amplitude of contraction induced by 80 mM K+in the absence of propofol was normalized as a relative force of 1.0 for each strip. [black circle], control;[white triangle], 10 [micro sign]M propofol;[white square], 30 [micro sign]M propofol;[black triangle down], 100 [micro sign]M propofol. Mean of data from six strips, with SD shown by vertical lines. *Significant difference from the corresponding control (P < 0.05).
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Figure 3. Concentration-dependent effect of propofol on contractions induced by various concentrations of norepinephrine. (A) Actual traces of contractions induced by various concentrations of norepinephrine in the absence (left panel) or presence (right panel) of 30 [micro sign]M propofol (both traces were obtained from a single smooth muscle strip). (B) Overlaid traces show the concentration-dependent effects of propofol on the contraction induced by 3 [micro sign]M norepinephrine (on an expanded time scale). (C) Summary of the effects of propofol on the maximum contractions induced by various concentrations of norepinephrine. The maximum amplitude of contraction induced by 3 [micro sign]M norepinephrine in the absence of propofol was normalized as a relative force of 1.0 for each strip. [black circle], control;[white triangle], 10 [micro sign]M propofol;[white square], 30 [micro sign]M propofol;[black triangle down], 100 [micro sign]M propofol. Mean of data from six strips, with SD shown by vertical lines. *Significant difference from the corresponding control (P < 0.05).
Figure 3. Concentration-dependent effect of propofol on contractions induced by various concentrations of norepinephrine. (A) Actual traces of contractions induced by various concentrations of norepinephrine in the absence (left panel) or presence (right panel) of 30 [micro sign]M propofol (both traces were obtained from a single smooth muscle strip). (B) Overlaid traces show the concentration-dependent effects of propofol on the contraction induced by 3 [micro sign]M norepinephrine (on an expanded time scale). (C) Summary of the effects of propofol on the maximum contractions induced by various concentrations of norepinephrine. The maximum amplitude of contraction induced by 3 [micro sign]M norepinephrine in the absence of propofol was normalized as a relative force of 1.0 for each strip. [black circle], control;[white triangle], 10 [micro sign]M propofol;[white square], 30 [micro sign]M propofol;[black triangle down], 100 [micro sign]M propofol. Mean of data from six strips, with SD shown by vertical lines. *Significant difference from the corresponding control (P < 0.05).
Figure 3. Concentration-dependent effect of propofol on contractions induced by various concentrations of norepinephrine. (A) Actual traces of contractions induced by various concentrations of norepinephrine in the absence (left panel) or presence (right panel) of 30 [micro sign]M propofol (both traces were obtained from a single smooth muscle strip). (B) Overlaid traces show the concentration-dependent effects of propofol on the contraction induced by 3 [micro sign]M norepinephrine (on an expanded time scale). (C) Summary of the effects of propofol on the maximum contractions induced by various concentrations of norepinephrine. The maximum amplitude of contraction induced by 3 [micro sign]M norepinephrine in the absence of propofol was normalized as a relative force of 1.0 for each strip. [black circle], control;[white triangle], 10 [micro sign]M propofol;[white square], 30 [micro sign]M propofol;[black triangle down], 100 [micro sign]M propofol. Mean of data from six strips, with SD shown by vertical lines. *Significant difference from the corresponding control (P < 0.05).
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Effect of Propofol on the Contractions Induced by Norepinephrine and Caffeine in Ca2+-freeSolution
The effect of propofol was examined for the contractions induced by successive applications of norepinephrine in Ca2+-freesolution (see Figure 1for the protocol). Propofol (30 and 100 [micro sign]M) significantly attenuated the contraction induced by the first norepinephrine, but enhanced the contraction induced by the second norepinephrine, with both effects occurring in a concentration-dependent manner (n = 5, P < 0.05;Figure 4).
Figure 4. Effect of propofol on contractions induced by norepinephrine in Ca2+-freesolution. (A) Overlaid traces of contractions induced by successively applied norepinephrine in Ca2+-freesolution containing 2 mM EGTA. Norepinephrine (3 [micro sign]M) was successively applied with a 2-min interval, as indicated by the bars. The first norepinephrine was applied for 0.5 min either in the absence (control) or presence (propofol treatment) or propofol, and the second norepinephrine was applied for 2 min with no further addition of propofol (i.e., in the absence of propofol). The results were obtained from a single smooth muscle strip. (B) Summary of the effects of propofol on contractions induced by successively applied norepinephrine in Ca2+-freesolution containing 2 mM EGTA. The first norepinephrine and second norepinephrine were applied as described in panel A. The maximum amplitude of contraction induced by the first norepinephrine in the absence of propofol was normalized as a relative force of 1.0 for each strip. Mean of data from five strips, with SD shown by vertical lines. *Significant difference from the corresponding control (P < 0.05).
Figure 4. Effect of propofol on contractions induced by norepinephrine in Ca2+-freesolution. (A) Overlaid traces of contractions induced by successively applied norepinephrine in Ca2+-freesolution containing 2 mM EGTA. Norepinephrine (3 [micro sign]M) was successively applied with a 2-min interval, as indicated by the bars. The first norepinephrine was applied for 0.5 min either in the absence (control) or presence (propofol treatment) or propofol, and the second norepinephrine was applied for 2 min with no further addition of propofol (i.e., in the absence of propofol). The results were obtained from a single smooth muscle strip. (B) Summary of the effects of propofol on contractions induced by successively applied norepinephrine in Ca2+-freesolution containing 2 mM EGTA. The first norepinephrine and second norepinephrine were applied as described in panel A. The maximum amplitude of contraction induced by the first norepinephrine in the absence of propofol was normalized as a relative force of 1.0 for each strip. Mean of data from five strips, with SD shown by vertical lines. *Significant difference from the corresponding control (P < 0.05).
Figure 4. Effect of propofol on contractions induced by norepinephrine in Ca2+-freesolution. (A) Overlaid traces of contractions induced by successively applied norepinephrine in Ca2+-freesolution containing 2 mM EGTA. Norepinephrine (3 [micro sign]M) was successively applied with a 2-min interval, as indicated by the bars. The first norepinephrine was applied for 0.5 min either in the absence (control) or presence (propofol treatment) or propofol, and the second norepinephrine was applied for 2 min with no further addition of propofol (i.e., in the absence of propofol). The results were obtained from a single smooth muscle strip. (B) Summary of the effects of propofol on contractions induced by successively applied norepinephrine in Ca2+-freesolution containing 2 mM EGTA. The first norepinephrine and second norepinephrine were applied as described in panel A. The maximum amplitude of contraction induced by the first norepinephrine in the absence of propofol was normalized as a relative force of 1.0 for each strip. Mean of data from five strips, with SD shown by vertical lines. *Significant difference from the corresponding control (P < 0.05).
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The amplitude of contraction induced by 10 mM caffeine in Ca2+-freesolution was 91 +/- 9% of that induced by 3 [micro sign]M norepinephrine under identical conditions (n = 4). Propofol (100 [micro sign]M) had no effect on the caffeine-induced contraction (1.09 +/- 0.09 times control, n = 4).
Effect of Propofol on Increases in [Ca2+]iand Force Induced by High K+and Norepinephrine in Ryanodine-treated Strips
High K+and norepinephrine each produced a large phasic followed by a tonic increase in [Ca2+]iand force (Figure 5A and Figure 5B, labeled “control”). After the application of ryanodine, the resting [Ca2+]iwas slightly increased (Table 1). In ryanodine-treated strips, norepinephrine failed to induce a phasic increase in either [Ca2+]ior force, each of which showed only a slowly developed tonic increase (Figure 5B, labeled “ryanodine-treated”).
Figure 5. Effect of ryanodine on increases in [Ca2+]i(upper records) and force (lower records) induced by high K+(A) or norepinephrine (B). Norepinephrine (3 [micro sign]M) and high K+(128 mM) were applied for 2-min periods with a 20-min interval (control). After the control responses had been recorded, ryanodine (50 [micro sign]M) with 10 mM caffeine was applied for 5 min, followed by a 10-min washout of caffeine. Norepinephrine and high K+were then applied (with a 20-min interval) in the presence of 10 [micro sign]M ryanodine. Thin and thick lines indicate the responses obtained before and after application of ryanodine, respectively. The results illustrated were obtained from a single smooth muscle strip and were reproducible in another three strips.
Figure 5. Effect of ryanodine on increases in [Ca2+]i(upper records) and force (lower records) induced by high K+(A) or norepinephrine (B). Norepinephrine (3 [micro sign]M) and high K+(128 mM) were applied for 2-min periods with a 20-min interval (control). After the control responses had been recorded, ryanodine (50 [micro sign]M) with 10 mM caffeine was applied for 5 min, followed by a 10-min washout of caffeine. Norepinephrine and high K+were then applied (with a 20-min interval) in the presence of 10 [micro sign]M ryanodine. Thin and thick lines indicate the responses obtained before and after application of ryanodine, respectively. The results illustrated were obtained from a single smooth muscle strip and were reproducible in another three strips.
Figure 5. Effect of ryanodine on increases in [Ca2+]i(upper records) and force (lower records) induced by high K+(A) or norepinephrine (B). Norepinephrine (3 [micro sign]M) and high K+(128 mM) were applied for 2-min periods with a 20-min interval (control). After the control responses had been recorded, ryanodine (50 [micro sign]M) with 10 mM caffeine was applied for 5 min, followed by a 10-min washout of caffeine. Norepinephrine and high K+were then applied (with a 20-min interval) in the presence of 10 [micro sign]M ryanodine. Thin and thick lines indicate the responses obtained before and after application of ryanodine, respectively. The results illustrated were obtained from a single smooth muscle strip and were reproducible in another three strips.
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Table 1. Effects of Ryanodine on Increases in [Ca2+]iand Force Induced by 128 mM K+or 3 [micro sign]M Norepinephrine (NE) in Smooth Muscle Strips of the Rabbit Mesenteric Artery 
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Table 1. Effects of Ryanodine on Increases in [Ca2+]iand Force Induced by 128 mM K+or 3 [micro sign]M Norepinephrine (NE) in Smooth Muscle Strips of the Rabbit Mesenteric Artery 
×
In ryanodine-treated strips, propofol increased the resting [Ca (2+)]iin a concentration-dependent manner, without increasing the resting force (Figure 6and Figure 7). In ryanodine-treated smooth muscle, the resting control [Ca2+]iwas 87.3 +/- 13.8 nM, whereas the corresponding values in the presence of 10 [micro sign]M, 30 [micro sign]M, and 100 [micro sign]M propofol were greater: 92.2 +/- 14.6 nM, 98.7 +/- 12.8 nM, and 100.9 +/- 7.2 nM (n = 4, P < 0.05). However, propofol did not increase the [Ca2+]iin Ca2+-freesolution in ryanodine-treated strips. For example, when propofol (100 [micro sign]M) was applied after a 2-min application of the Ca2+-freesolution, the resting values obtained for [Ca2+]idid not differ before (56.2 +/- 12.5 nM) and after (57.6 +/- 13.9 nM) a 2-min application of propofol (n = 4, P > 0.05).
Figure 6. Concentration-dependent effects of propofol on increases in [Ca (2+)]iand force induced by 128 mM K+in ryanodine-treated smooth muscle strips. Actual traces of simultaneous measurements of increases in [Ca (2+)]i(Aa) and force (Ba) induced by high K+(both sets of traces were obtained from a single smooth muscle strip). High K+was applied for 2 min at 20-min intervals in the presence or absence of propofol. Propofol was applied for 5 min before and was present during the subsequent application of 128 mM K+. Right-hand panels show concentration-dependent effects of propofol on increases in [Ca2+]i(Ab) and force (Bb) induced by 128 mM K+. Because propofol increased the resting [Ca2+](i), the delta [Ca2+]ivalues (delta [Ca2+]i= the maximum [Ca2+]ilevel reached resting [Ca2+]i) induced by 128 mM K+were calculated in the presence and absence of propofol, and then the percentage inhibition induced by propofol was calculated (Ab). In (Bb), propofol-induced changes in the maximum force induced by 128 mM K+are expressed as percentages of the corresponding values in the absence of propofol. Mean of data from four strips, with SD shown by vertical lines. *Significant difference from the corresponding control (P < 0.05).
Figure 6. Concentration-dependent effects of propofol on increases in [Ca (2+)]iand force induced by 128 mM K+in ryanodine-treated smooth muscle strips. Actual traces of simultaneous measurements of increases in [Ca (2+)]i(Aa) and force (Ba) induced by high K+(both sets of traces were obtained from a single smooth muscle strip). High K+was applied for 2 min at 20-min intervals in the presence or absence of propofol. Propofol was applied for 5 min before and was present during the subsequent application of 128 mM K+. Right-hand panels show concentration-dependent effects of propofol on increases in [Ca2+]i(Ab) and force (Bb) induced by 128 mM K+. Because propofol increased the resting [Ca2+](i), the delta [Ca2+]ivalues (delta [Ca2+]i= the maximum [Ca2+]ilevel reached resting [Ca2+]i) induced by 128 mM K+were calculated in the presence and absence of propofol, and then the percentage inhibition induced by propofol was calculated (Ab). In (Bb), propofol-induced changes in the maximum force induced by 128 mM K+are expressed as percentages of the corresponding values in the absence of propofol. Mean of data from four strips, with SD shown by vertical lines. *Significant difference from the corresponding control (P < 0.05).
Figure 6. Concentration-dependent effects of propofol on increases in [Ca (2+)]iand force induced by 128 mM K+in ryanodine-treated smooth muscle strips. Actual traces of simultaneous measurements of increases in [Ca (2+)]i(Aa) and force (Ba) induced by high K+(both sets of traces were obtained from a single smooth muscle strip). High K+was applied for 2 min at 20-min intervals in the presence or absence of propofol. Propofol was applied for 5 min before and was present during the subsequent application of 128 mM K+. Right-hand panels show concentration-dependent effects of propofol on increases in [Ca2+]i(Ab) and force (Bb) induced by 128 mM K+. Because propofol increased the resting [Ca2+](i), the delta [Ca2+]ivalues (delta [Ca2+]i= the maximum [Ca2+]ilevel reached resting [Ca2+]i) induced by 128 mM K+were calculated in the presence and absence of propofol, and then the percentage inhibition induced by propofol was calculated (Ab). In (Bb), propofol-induced changes in the maximum force induced by 128 mM K+are expressed as percentages of the corresponding values in the absence of propofol. Mean of data from four strips, with SD shown by vertical lines. *Significant difference from the corresponding control (P < 0.05).
×
Figure 7. Concentration-dependent effects of propofol on increases in [Ca (2+)]iand force induced by norepinephrine in ryanodine-treated smooth muscle strips. Actual traces of simultaneous measurements of increases in [Ca (2+)]i(Aa) and force (Ba) induced by 3 [micro sign]M norepinephrine (both sets of traces were obtained from a single smooth muscle strip). Norepinephrine was applied for 2 min at 20-min intervals in the presence or absence of propofol. Propofol was applied for 5 min before and was present during the subsequent application of norepinephrine. Right-hand panels show concentration-dependent effects of propofol on increases in [Ca2+]i(Ab) and force (Bb) induced by norepinephrine. The delta [Ca2+]ivalues (see legend to Figure 6) induced by 3 [micro sign]M norepinephrine were calculated in the presence and absence of propofol, and then the percentage inhibition induced by propofol was calculated (Ab). In (Bb), propofol-induced changes in the maximum force induced by 3 [micro sign]M norepinephrine are expressed as percentages of the corresponding control (in the absence of propofol). Mean of data from four strips, with SD shown by vertical lines. *Significant difference from the corresponding control (P < 0.05).
Figure 7. Concentration-dependent effects of propofol on increases in [Ca (2+)]iand force induced by norepinephrine in ryanodine-treated smooth muscle strips. Actual traces of simultaneous measurements of increases in [Ca (2+)]i(Aa) and force (Ba) induced by 3 [micro sign]M norepinephrine (both sets of traces were obtained from a single smooth muscle strip). Norepinephrine was applied for 2 min at 20-min intervals in the presence or absence of propofol. Propofol was applied for 5 min before and was present during the subsequent application of norepinephrine. Right-hand panels show concentration-dependent effects of propofol on increases in [Ca2+]i(Ab) and force (Bb) induced by norepinephrine. The delta [Ca2+]ivalues (see legend to Figure 6) induced by 3 [micro sign]M norepinephrine were calculated in the presence and absence of propofol, and then the percentage inhibition induced by propofol was calculated (Ab). In (Bb), propofol-induced changes in the maximum force induced by 3 [micro sign]M norepinephrine are expressed as percentages of the corresponding control (in the absence of propofol). Mean of data from four strips, with SD shown by vertical lines. *Significant difference from the corresponding control (P < 0.05).
Figure 7. Concentration-dependent effects of propofol on increases in [Ca (2+)]iand force induced by norepinephrine in ryanodine-treated smooth muscle strips. Actual traces of simultaneous measurements of increases in [Ca (2+)]i(Aa) and force (Ba) induced by 3 [micro sign]M norepinephrine (both sets of traces were obtained from a single smooth muscle strip). Norepinephrine was applied for 2 min at 20-min intervals in the presence or absence of propofol. Propofol was applied for 5 min before and was present during the subsequent application of norepinephrine. Right-hand panels show concentration-dependent effects of propofol on increases in [Ca2+]i(Ab) and force (Bb) induced by norepinephrine. The delta [Ca2+]ivalues (see legend to Figure 6) induced by 3 [micro sign]M norepinephrine were calculated in the presence and absence of propofol, and then the percentage inhibition induced by propofol was calculated (Ab). In (Bb), propofol-induced changes in the maximum force induced by 3 [micro sign]M norepinephrine are expressed as percentages of the corresponding control (in the absence of propofol). Mean of data from four strips, with SD shown by vertical lines. *Significant difference from the corresponding control (P < 0.05).
×
In ryanodine-treated strips, propofol significantly attenuated the maximum increases in [Ca2+]iand force induced by high K+(Figure 6) and norepinephrine (Figure 7).
Effect of Propofol on Norepinephrine-induced Increases in [Ca (2+)]iand Force in the Presence of Nicardipine in Ryanodine-treated Strips
In ryanodine-treated strips, nicardipine slightly decreased the resting [Ca2+]i(from 104 +/- 190 nM to 92.6 +/- 10.8 nM; n = 4, P < 0.05) and blocked the increases in [Ca2+]iand force induced by high K+(Figure 8A). Nicardipine also attenuated the increases in [Ca (2+)]i(peak level reduced from 168.4 +/- 38 nM to 100.4 +/- 14.4 nM) and force (from 144 +/- 42 [micro sign]N to 8.3 +/- 3.2 [micro sign]N) that were induced by norepinephrine (P < 0.05, n = 4;Figure 8B).
Figure 8. Effect of propofol on increases in [Ca2+]iand force induced by high K+(A) or norepinephrine (B) in the presence of nicardipine in a ryanodine-treated smooth muscle strip. After the muscle strip had been treated with ryanodine (as described for Figure 5), high K (+)(128 mM, A) and norepinephrine (3 [micro sign]M, B) were applied successively for 2-min periods with a 10-min interval (left panels). This protocol was repeated after a 20-min intermission. Nicardipine (0.3 [micro sign]M) was then pretreated for 5 min and it was present throughout the rest of the experiment. High K+and norepinephrine were again successively applied for 2 min in the presence of nicardipine (middle panels), followed by a washout for 20 min. Propofol (100 [micro sign]M) was pretreated for 2 min and it was present during the subsequent application of either high K+or norepinephrine (right panels). The results illustrated were obtained from a single smooth muscle strip and were reproducible in another three strips.
Figure 8. Effect of propofol on increases in [Ca2+]iand force induced by high K+(A) or norepinephrine (B) in the presence of nicardipine in a ryanodine-treated smooth muscle strip. After the muscle strip had been treated with ryanodine (as described for Figure 5), high K (+)(128 mM, A) and norepinephrine (3 [micro sign]M, B) were applied successively for 2-min periods with a 10-min interval (left panels). This protocol was repeated after a 20-min intermission. Nicardipine (0.3 [micro sign]M) was then pretreated for 5 min and it was present throughout the rest of the experiment. High K+and norepinephrine were again successively applied for 2 min in the presence of nicardipine (middle panels), followed by a washout for 20 min. Propofol (100 [micro sign]M) was pretreated for 2 min and it was present during the subsequent application of either high K+or norepinephrine (right panels). The results illustrated were obtained from a single smooth muscle strip and were reproducible in another three strips.
Figure 8. Effect of propofol on increases in [Ca2+]iand force induced by high K+(A) or norepinephrine (B) in the presence of nicardipine in a ryanodine-treated smooth muscle strip. After the muscle strip had been treated with ryanodine (as described for Figure 5), high K (+)(128 mM, A) and norepinephrine (3 [micro sign]M, B) were applied successively for 2-min periods with a 10-min interval (left panels). This protocol was repeated after a 20-min intermission. Nicardipine (0.3 [micro sign]M) was then pretreated for 5 min and it was present throughout the rest of the experiment. High K+and norepinephrine were again successively applied for 2 min in the presence of nicardipine (middle panels), followed by a washout for 20 min. Propofol (100 [micro sign]M) was pretreated for 2 min and it was present during the subsequent application of either high K+or norepinephrine (right panels). The results illustrated were obtained from a single smooth muscle strip and were reproducible in another three strips.
×
In the presence of ryanodine with nicardipine, propofol increased the resting [Ca2+]i(from 92.6 +/- 108 nM to 111.3 +/- 13.2 nM) without increasing the resting force (n = 4, P < 0.05;Figure 8). In the continued presence of nicardipine, high K+then partially reversed the increase in the resting [Ca2+]iinduced by propofol (from 111.5 +/- 5.8 nM to 98.2 +/- 3.7 nM, n = 4;Figure 8A). In contrast, norepinephrine further increased both [Ca2+]iand force in the presence of nicardipine together with propofol. In the presence of propofol, the levels of [Ca2+]i(123.5 +/- 22.8 nM) and force (18 +/- 6.9 [micro sign]N) reached when norepinephrine was applied were significantly larger (Figure 8B) than those reached in the absence of propofol (for [Ca2+]i, 100.4 +/- 15 nM; for force, 8.3 +/- 3.2 [micro sign]N; n = 4, P < 0.05), with both tests performed in the presence of nicardipine. However, when the delta [Ca2+]ivalues (see the legend to Figure 6) were calculated in the presence of nicardipine, the responses to norepinephrine of 9.8 +/- 1.8 nM and 13.2 +/- 3 nM in the absence and presence of propofol, respectively, did not differ (n = 4, P > 0.05).
Discussion
In the present experiments, on the endothelium-denuded rabbit mesenteric resistance artery, norepinephrine produced a phasic followed by a tonic increase in both [Ca2+]iand force in untreated smooth muscle strips, although this agonist failed to produce a phasic increase in [Ca2+]iin ryanodine-treated strips and produced only a monotonic response (Figure 5). Because the residual increase in [Ca2+]iinduced by norepinephrine in ryanodine-treated strips was completely abolished in Ca2+-freesolution, [18–20,22] these results confirm that in ryanodine-treated smooth muscle, norepinephrine increases [Ca2+]ionly through an increase in Ca2+influx.
Effect of Propofol on Norepinephrine-activated Ca2+Influx
In the present experiments, nicardipine (0.3 [micro sign]M) completely blocked the high K+-inducedincreases in [Ca2+]iand force in ryanodine-treated smooth muscle strips. In contrast, the same treatment markedly, but not completely, attenuated the norepinephrine-induced increases in [Ca2+]iand force in ryanodine-treated strips, which suggest that norepinephrine-induced contraction is due to Ca2+influx through both dihydropyridine-sensitive and -insensitive Ca2+channels. [20,22] In ryanodine-treated strips, propofol similarly attenuated the high K+-inducedincreases in [Ca2+]iand force (Figure 6) and partly attenuated the norepinephrine-induced increases in [Ca2+]iand force (Figure 7). In the presence of nicardipine, propofol (100 [micro sign]M) had no additional effect on the remaining norepinephrine-induced increases in [Ca2+]iand force (Figure 8). Investigators have found in the rat thoracic aorta that propofol inhibits Ca (2+-induced) contractions in Ca2+-freesolution containing high K+. [10] Furthermore, propofol inhibited the Ca2+entry through L-type Ca2+channels that is activated by endothelin-1 in cultured A 10 and rat aortic smooth muscle cells. [27] These results suggest that in smooth muscle of the rabbit mesenteric resistance artery, propofol inhibits Ca2+influx through the dihydropyridine-sensitive, L-type Ca2+channel, and by this means attenuates the contractions induced by high K+and norepinephrine.
In the present experiments, propofol exerted a more potent attenuating effect on the contraction induced by high K+than on that induced by the [Greek small letter alpha]-receptor agonist, norepinephrine. A similar observation was already made in the rat thoracic aorta. [10] In contrast to these findings, it was found in the porcine coronary artery that the relaxation induced by propofol was more pronounced in preparations contracted by agonists than in those contracted by high K+. [12] In the same study, investigators also found that in the presence of nifedipine, propofol attenuated the Ca2+-inducedcontraction in the presence of norepinephrine, suggesting that there might be an important effect of propofol on Ca2+influx through receptor-mediated mechanisms in the porcine coronary artery. Species and vascular heterogeneity may account for these differences.
Effect of Propofol on Norepinephrine-activated Ca2+Release
Propofol attenuated the phasic increase in force induced by norepinephrine in the presence (Figure 3) and in absence of extracellular Ca2+(Figure 4). However, at the same concentration, propofol had no effect on the caffeine-induced contraction in Ca2+-freesolution. These results suggest that propofol selectively inhibits the mechanism involved in the norepinephrine-induced Ca2+release from the storage sites. This conclusion is supported by the present finding that, when norepinephrine was applied twice in succession in Ca2+-freesolution, with propofol being given only during the first application, propofol enhanced the contraction induced by the second norepinephrine even though it had attenuated the contraction induced by the first norepinephrine (Figure 4). This result can be explained as follows. In Ca2+-freesolution, a portion of the stored Ca2+is released during the first application of norepinephrine and contributes to the first norepinephrine-contraction, whereas the remaining, smaller amount of stored Ca2+is responsible for the contraction induced by the second application of norepinephrine. Propofol inhibits the Ca2+release induced by the first application of norepinephrine and thus increases the amount of stored Ca2+that remains within the storage sites. As a result, more Ca2+is available for release, thus enhancing the contraction induced by the second application of norepinephrine. It was found previously that, in cultured A 10 and rat aortic smooth muscle cells, propofol inhibits the synthesis of inositol (1,4,5)-triphosphate (InsP3) induced by agonists, suggesting that propofol may indirectly reduce the release of Ca2+from the InsP3-sensitiveintracellular pool. [27] Together these results suggest that the inhibitory action of propofol on norepinephrine-induced Ca2+release is most likely caused by an inhibitory action on the norepinephrine-induced synthesis of InsP3rather than on the InsP3-inducedCa2+release mechanism itself. However, this hypothesis remains to be clarified in future investigations.
Effect of Propofol on Resting [Ca2+]i
In ryanodine-treated strips, propofol increased the resting [Ca (2+)]iwithout producing an increase in force. The propofol-induced increase in [Ca2+]iwas not modified by nicardipine, but it was attenuated by high K+and abolished by an application of Ca2+-freesolution, suggesting that it is due to an activation of voltage-insensitive Ca2+influx. In has been suggested that in some smooth muscle cells, Ca (2+) influxes can be evoked by agonists through an activation of both the dihydropyridine-sensitive and -insensitive pathways. [28–30] One of the latter type might be the nonselective cation channel, which has been found to be voltage insensitive [28,30,31]; indeed, membrane depolarization actually causes an inhibition of this channel's activity. Alternatively, propofol may decrease the rate of Ca2+elimination from the cell and thereby increase the resting [Ca2+]i. Because in ryanodine-treated cells, Ca2+storage sites could be leaky, a modest inhibition of Ca2+elimination pathways (such as Na+/Ca2+exchange and sarcolemmal Ca2+ATPase) might lead to a greater increase in [Ca2+]ithan it would in non-ryanodine-treated cells. In preliminary experiments, we found that in Ca2+-free solution containing 2 mM EGTA, the rate of decrease (but not the rate of increase) in the [Ca2+]iresponse evoked by an application of caffeine in Ca2+-freesolution was inhibited by 100 [micro sign]M propofol (-16 +/- 0.4 nM [Ca2+]i/sin control and -10.3 +/- 0.4 nM [Ca2+]i/sin the presence of propofol; n = 3, P < 0.05). Propofol slightly increased the resting [Ca2+]iin non-ryanodine-treated cells, but the effect was significantly weaker than that seen in ryanodine-treated cells (n = 4, P < 0.05). These findings support the hypothesis that propofol decreases the rate of Ca2+elimination from the cell. However, based on the results of our current experiments, we cannot be certain which mechanisms contribute to the propofol-induced increase in resting [Ca2+]iin ryanodine-treated cells.
Propofol increased the resting [Ca2+]i, which further increased the level of [Ca2+]ireached in the presence of norepinephrine, thus enhancing the norepinephrine-induced force seen in the presence of nicardipine. This suggests that the vasorelaxant effect of propofol on a norepinephrine-induced increase in force could be somewhat counteracted by its action on Ca2+handling mechanisms. In contrast to our current findings, it has been found in cultured A 10 and rat aortic smooth muscle cells that propofol alone does not change basal [Ca2+]iin cells perfused in the presence or absence of extracellular Ca2+. [27] Thus there may be species and cell-type differences in this action of propofol. Furthermore, because these experiments were performed at room temperature, it is possible that the alteration in temperature may change the balance between activation of sarcoplasmic reticulum Ca2+release, Ca (2+) entry, Ca2+elimination, and Ca2+uptake into the internal stores. This alteration in balance might result in some quantitative difference in behavior at normothermia, although it should make little qualitative difference in the behavior. This potential complicating factor should be noted.
Norepinephrine enhances the myofilament Ca2+sensitivity in smooth muscle of the rabbit mesenteric artery. [19–22] It is consistent with this previous finding that, despite a far lower [Ca2+]i, more force was developed with norepinephrine than with high K+in ryanodinetreated muscle strips (Figure 5, Figure 6, Figure 7Figure 8). In the current experiments, although propofol slightly increased the resting [Ca2+]i, no force was developed (Figure 8), suggesting that unlike norepinephrine, propofol may not modify the myofilament Ca2+sensitivity.
Based on the assumption that the plasma protein binding for propofol is 97% or 98%, the concentrations (10 - 100 [micro sign]M) of propofol that we used may be supratherapeutic. However, the situation is not straightforward, because the microkinetic behavior of propofol within the vascular space has not been properly characterized. [9] Furthermore, it is not known whether only unbound propofol possesses vasomotor activity or whether any bound fraction may also have a vasomotor effect. [9] 
In conclusion, in smooth muscle of the rabbit mesenteric resistance artery, propofol attenuates the norepinephrine-induced contraction through an inhibition of dihydropyridine-sensitive Ca2+influx and of Ca (2+) release from the storage sites. Propofol also increases resting [Ca (2+)]i, possibly as a result of an inhibition of [Ca2+]iremoval mechanisms. Because our study was done using endothelium-denuded strips, we cannot draw any conclusions about the possible influence of the endothelium on the actions of propofol in small mesenteric resistance arteries. However, our results suggest that the different actions of propofol on Ca2+handling mechanisms in smooth muscle cells may partly account for the variety of effects on agonist-induced contractions seen with propofol in various types of vascular smooth muscle.
The authors thank Dr. R. J. Timms for critical reading of the manuscript and T. Kamiya and J. Kajikuri for their technical assistance. Propofol was a gift from Zeneca Pharmaceuticals (Mereside, Macclesfield, UK).
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Figure 1. Experimental protocol used to investigate the action of propofol on norepinephrine-induced contraction in Ca2+-freesolution (see Materials and Methods for further details).
Figure 1. Experimental protocol used to investigate the action of propofol on norepinephrine-induced contraction in Ca2+-freesolution (see Materials and Methods for further details).
Figure 1. Experimental protocol used to investigate the action of propofol on norepinephrine-induced contraction in Ca2+-freesolution (see Materials and Methods for further details).
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Figure 2. Concentration-dependent effect of propofol on contractions induced by various concentrations of high K+. (A) Actual traces of contractions induced by various concentrations of high K+in the absence (left panel) or presence (right panel) of 30 [micro sign]M propofol (both traces were obtained from a single smooth muscle strip). (B) Concentration dependence of the effect of propofol on the maximum (phasic) contractions induced by various concentrations of high K+. The maximum amplitude of contraction induced by 80 mM K+in the absence of propofol was normalized as a relative force of 1.0 for each strip. [black circle], control;[white triangle], 10 [micro sign]M propofol;[white square], 30 [micro sign]M propofol;[black triangle down], 100 [micro sign]M propofol. Mean of data from six strips, with SD shown by vertical lines. *Significant difference from the corresponding control (P < 0.05).
Figure 2. Concentration-dependent effect of propofol on contractions induced by various concentrations of high K+. (A) Actual traces of contractions induced by various concentrations of high K+in the absence (left panel) or presence (right panel) of 30 [micro sign]M propofol (both traces were obtained from a single smooth muscle strip). (B) Concentration dependence of the effect of propofol on the maximum (phasic) contractions induced by various concentrations of high K+. The maximum amplitude of contraction induced by 80 mM K+in the absence of propofol was normalized as a relative force of 1.0 for each strip. [black circle], control;[white triangle], 10 [micro sign]M propofol;[white square], 30 [micro sign]M propofol;[black triangle down], 100 [micro sign]M propofol. Mean of data from six strips, with SD shown by vertical lines. *Significant difference from the corresponding control (P < 0.05).
Figure 2. Concentration-dependent effect of propofol on contractions induced by various concentrations of high K+. (A) Actual traces of contractions induced by various concentrations of high K+in the absence (left panel) or presence (right panel) of 30 [micro sign]M propofol (both traces were obtained from a single smooth muscle strip). (B) Concentration dependence of the effect of propofol on the maximum (phasic) contractions induced by various concentrations of high K+. The maximum amplitude of contraction induced by 80 mM K+in the absence of propofol was normalized as a relative force of 1.0 for each strip. [black circle], control;[white triangle], 10 [micro sign]M propofol;[white square], 30 [micro sign]M propofol;[black triangle down], 100 [micro sign]M propofol. Mean of data from six strips, with SD shown by vertical lines. *Significant difference from the corresponding control (P < 0.05).
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Figure 3. Concentration-dependent effect of propofol on contractions induced by various concentrations of norepinephrine. (A) Actual traces of contractions induced by various concentrations of norepinephrine in the absence (left panel) or presence (right panel) of 30 [micro sign]M propofol (both traces were obtained from a single smooth muscle strip). (B) Overlaid traces show the concentration-dependent effects of propofol on the contraction induced by 3 [micro sign]M norepinephrine (on an expanded time scale). (C) Summary of the effects of propofol on the maximum contractions induced by various concentrations of norepinephrine. The maximum amplitude of contraction induced by 3 [micro sign]M norepinephrine in the absence of propofol was normalized as a relative force of 1.0 for each strip. [black circle], control;[white triangle], 10 [micro sign]M propofol;[white square], 30 [micro sign]M propofol;[black triangle down], 100 [micro sign]M propofol. Mean of data from six strips, with SD shown by vertical lines. *Significant difference from the corresponding control (P < 0.05).
Figure 3. Concentration-dependent effect of propofol on contractions induced by various concentrations of norepinephrine. (A) Actual traces of contractions induced by various concentrations of norepinephrine in the absence (left panel) or presence (right panel) of 30 [micro sign]M propofol (both traces were obtained from a single smooth muscle strip). (B) Overlaid traces show the concentration-dependent effects of propofol on the contraction induced by 3 [micro sign]M norepinephrine (on an expanded time scale). (C) Summary of the effects of propofol on the maximum contractions induced by various concentrations of norepinephrine. The maximum amplitude of contraction induced by 3 [micro sign]M norepinephrine in the absence of propofol was normalized as a relative force of 1.0 for each strip. [black circle], control;[white triangle], 10 [micro sign]M propofol;[white square], 30 [micro sign]M propofol;[black triangle down], 100 [micro sign]M propofol. Mean of data from six strips, with SD shown by vertical lines. *Significant difference from the corresponding control (P < 0.05).
Figure 3. Concentration-dependent effect of propofol on contractions induced by various concentrations of norepinephrine. (A) Actual traces of contractions induced by various concentrations of norepinephrine in the absence (left panel) or presence (right panel) of 30 [micro sign]M propofol (both traces were obtained from a single smooth muscle strip). (B) Overlaid traces show the concentration-dependent effects of propofol on the contraction induced by 3 [micro sign]M norepinephrine (on an expanded time scale). (C) Summary of the effects of propofol on the maximum contractions induced by various concentrations of norepinephrine. The maximum amplitude of contraction induced by 3 [micro sign]M norepinephrine in the absence of propofol was normalized as a relative force of 1.0 for each strip. [black circle], control;[white triangle], 10 [micro sign]M propofol;[white square], 30 [micro sign]M propofol;[black triangle down], 100 [micro sign]M propofol. Mean of data from six strips, with SD shown by vertical lines. *Significant difference from the corresponding control (P < 0.05).
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Figure 4. Effect of propofol on contractions induced by norepinephrine in Ca2+-freesolution. (A) Overlaid traces of contractions induced by successively applied norepinephrine in Ca2+-freesolution containing 2 mM EGTA. Norepinephrine (3 [micro sign]M) was successively applied with a 2-min interval, as indicated by the bars. The first norepinephrine was applied for 0.5 min either in the absence (control) or presence (propofol treatment) or propofol, and the second norepinephrine was applied for 2 min with no further addition of propofol (i.e., in the absence of propofol). The results were obtained from a single smooth muscle strip. (B) Summary of the effects of propofol on contractions induced by successively applied norepinephrine in Ca2+-freesolution containing 2 mM EGTA. The first norepinephrine and second norepinephrine were applied as described in panel A. The maximum amplitude of contraction induced by the first norepinephrine in the absence of propofol was normalized as a relative force of 1.0 for each strip. Mean of data from five strips, with SD shown by vertical lines. *Significant difference from the corresponding control (P < 0.05).
Figure 4. Effect of propofol on contractions induced by norepinephrine in Ca2+-freesolution. (A) Overlaid traces of contractions induced by successively applied norepinephrine in Ca2+-freesolution containing 2 mM EGTA. Norepinephrine (3 [micro sign]M) was successively applied with a 2-min interval, as indicated by the bars. The first norepinephrine was applied for 0.5 min either in the absence (control) or presence (propofol treatment) or propofol, and the second norepinephrine was applied for 2 min with no further addition of propofol (i.e., in the absence of propofol). The results were obtained from a single smooth muscle strip. (B) Summary of the effects of propofol on contractions induced by successively applied norepinephrine in Ca2+-freesolution containing 2 mM EGTA. The first norepinephrine and second norepinephrine were applied as described in panel A. The maximum amplitude of contraction induced by the first norepinephrine in the absence of propofol was normalized as a relative force of 1.0 for each strip. Mean of data from five strips, with SD shown by vertical lines. *Significant difference from the corresponding control (P < 0.05).
Figure 4. Effect of propofol on contractions induced by norepinephrine in Ca2+-freesolution. (A) Overlaid traces of contractions induced by successively applied norepinephrine in Ca2+-freesolution containing 2 mM EGTA. Norepinephrine (3 [micro sign]M) was successively applied with a 2-min interval, as indicated by the bars. The first norepinephrine was applied for 0.5 min either in the absence (control) or presence (propofol treatment) or propofol, and the second norepinephrine was applied for 2 min with no further addition of propofol (i.e., in the absence of propofol). The results were obtained from a single smooth muscle strip. (B) Summary of the effects of propofol on contractions induced by successively applied norepinephrine in Ca2+-freesolution containing 2 mM EGTA. The first norepinephrine and second norepinephrine were applied as described in panel A. The maximum amplitude of contraction induced by the first norepinephrine in the absence of propofol was normalized as a relative force of 1.0 for each strip. Mean of data from five strips, with SD shown by vertical lines. *Significant difference from the corresponding control (P < 0.05).
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Figure 5. Effect of ryanodine on increases in [Ca2+]i(upper records) and force (lower records) induced by high K+(A) or norepinephrine (B). Norepinephrine (3 [micro sign]M) and high K+(128 mM) were applied for 2-min periods with a 20-min interval (control). After the control responses had been recorded, ryanodine (50 [micro sign]M) with 10 mM caffeine was applied for 5 min, followed by a 10-min washout of caffeine. Norepinephrine and high K+were then applied (with a 20-min interval) in the presence of 10 [micro sign]M ryanodine. Thin and thick lines indicate the responses obtained before and after application of ryanodine, respectively. The results illustrated were obtained from a single smooth muscle strip and were reproducible in another three strips.
Figure 5. Effect of ryanodine on increases in [Ca2+]i(upper records) and force (lower records) induced by high K+(A) or norepinephrine (B). Norepinephrine (3 [micro sign]M) and high K+(128 mM) were applied for 2-min periods with a 20-min interval (control). After the control responses had been recorded, ryanodine (50 [micro sign]M) with 10 mM caffeine was applied for 5 min, followed by a 10-min washout of caffeine. Norepinephrine and high K+were then applied (with a 20-min interval) in the presence of 10 [micro sign]M ryanodine. Thin and thick lines indicate the responses obtained before and after application of ryanodine, respectively. The results illustrated were obtained from a single smooth muscle strip and were reproducible in another three strips.
Figure 5. Effect of ryanodine on increases in [Ca2+]i(upper records) and force (lower records) induced by high K+(A) or norepinephrine (B). Norepinephrine (3 [micro sign]M) and high K+(128 mM) were applied for 2-min periods with a 20-min interval (control). After the control responses had been recorded, ryanodine (50 [micro sign]M) with 10 mM caffeine was applied for 5 min, followed by a 10-min washout of caffeine. Norepinephrine and high K+were then applied (with a 20-min interval) in the presence of 10 [micro sign]M ryanodine. Thin and thick lines indicate the responses obtained before and after application of ryanodine, respectively. The results illustrated were obtained from a single smooth muscle strip and were reproducible in another three strips.
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Figure 6. Concentration-dependent effects of propofol on increases in [Ca (2+)]iand force induced by 128 mM K+in ryanodine-treated smooth muscle strips. Actual traces of simultaneous measurements of increases in [Ca (2+)]i(Aa) and force (Ba) induced by high K+(both sets of traces were obtained from a single smooth muscle strip). High K+was applied for 2 min at 20-min intervals in the presence or absence of propofol. Propofol was applied for 5 min before and was present during the subsequent application of 128 mM K+. Right-hand panels show concentration-dependent effects of propofol on increases in [Ca2+]i(Ab) and force (Bb) induced by 128 mM K+. Because propofol increased the resting [Ca2+](i), the delta [Ca2+]ivalues (delta [Ca2+]i= the maximum [Ca2+]ilevel reached resting [Ca2+]i) induced by 128 mM K+were calculated in the presence and absence of propofol, and then the percentage inhibition induced by propofol was calculated (Ab). In (Bb), propofol-induced changes in the maximum force induced by 128 mM K+are expressed as percentages of the corresponding values in the absence of propofol. Mean of data from four strips, with SD shown by vertical lines. *Significant difference from the corresponding control (P < 0.05).
Figure 6. Concentration-dependent effects of propofol on increases in [Ca (2+)]iand force induced by 128 mM K+in ryanodine-treated smooth muscle strips. Actual traces of simultaneous measurements of increases in [Ca (2+)]i(Aa) and force (Ba) induced by high K+(both sets of traces were obtained from a single smooth muscle strip). High K+was applied for 2 min at 20-min intervals in the presence or absence of propofol. Propofol was applied for 5 min before and was present during the subsequent application of 128 mM K+. Right-hand panels show concentration-dependent effects of propofol on increases in [Ca2+]i(Ab) and force (Bb) induced by 128 mM K+. Because propofol increased the resting [Ca2+](i), the delta [Ca2+]ivalues (delta [Ca2+]i= the maximum [Ca2+]ilevel reached resting [Ca2+]i) induced by 128 mM K+were calculated in the presence and absence of propofol, and then the percentage inhibition induced by propofol was calculated (Ab). In (Bb), propofol-induced changes in the maximum force induced by 128 mM K+are expressed as percentages of the corresponding values in the absence of propofol. Mean of data from four strips, with SD shown by vertical lines. *Significant difference from the corresponding control (P < 0.05).
Figure 6. Concentration-dependent effects of propofol on increases in [Ca (2+)]iand force induced by 128 mM K+in ryanodine-treated smooth muscle strips. Actual traces of simultaneous measurements of increases in [Ca (2+)]i(Aa) and force (Ba) induced by high K+(both sets of traces were obtained from a single smooth muscle strip). High K+was applied for 2 min at 20-min intervals in the presence or absence of propofol. Propofol was applied for 5 min before and was present during the subsequent application of 128 mM K+. Right-hand panels show concentration-dependent effects of propofol on increases in [Ca2+]i(Ab) and force (Bb) induced by 128 mM K+. Because propofol increased the resting [Ca2+](i), the delta [Ca2+]ivalues (delta [Ca2+]i= the maximum [Ca2+]ilevel reached resting [Ca2+]i) induced by 128 mM K+were calculated in the presence and absence of propofol, and then the percentage inhibition induced by propofol was calculated (Ab). In (Bb), propofol-induced changes in the maximum force induced by 128 mM K+are expressed as percentages of the corresponding values in the absence of propofol. Mean of data from four strips, with SD shown by vertical lines. *Significant difference from the corresponding control (P < 0.05).
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Figure 7. Concentration-dependent effects of propofol on increases in [Ca (2+)]iand force induced by norepinephrine in ryanodine-treated smooth muscle strips. Actual traces of simultaneous measurements of increases in [Ca (2+)]i(Aa) and force (Ba) induced by 3 [micro sign]M norepinephrine (both sets of traces were obtained from a single smooth muscle strip). Norepinephrine was applied for 2 min at 20-min intervals in the presence or absence of propofol. Propofol was applied for 5 min before and was present during the subsequent application of norepinephrine. Right-hand panels show concentration-dependent effects of propofol on increases in [Ca2+]i(Ab) and force (Bb) induced by norepinephrine. The delta [Ca2+]ivalues (see legend to Figure 6) induced by 3 [micro sign]M norepinephrine were calculated in the presence and absence of propofol, and then the percentage inhibition induced by propofol was calculated (Ab). In (Bb), propofol-induced changes in the maximum force induced by 3 [micro sign]M norepinephrine are expressed as percentages of the corresponding control (in the absence of propofol). Mean of data from four strips, with SD shown by vertical lines. *Significant difference from the corresponding control (P < 0.05).
Figure 7. Concentration-dependent effects of propofol on increases in [Ca (2+)]iand force induced by norepinephrine in ryanodine-treated smooth muscle strips. Actual traces of simultaneous measurements of increases in [Ca (2+)]i(Aa) and force (Ba) induced by 3 [micro sign]M norepinephrine (both sets of traces were obtained from a single smooth muscle strip). Norepinephrine was applied for 2 min at 20-min intervals in the presence or absence of propofol. Propofol was applied for 5 min before and was present during the subsequent application of norepinephrine. Right-hand panels show concentration-dependent effects of propofol on increases in [Ca2+]i(Ab) and force (Bb) induced by norepinephrine. The delta [Ca2+]ivalues (see legend to Figure 6) induced by 3 [micro sign]M norepinephrine were calculated in the presence and absence of propofol, and then the percentage inhibition induced by propofol was calculated (Ab). In (Bb), propofol-induced changes in the maximum force induced by 3 [micro sign]M norepinephrine are expressed as percentages of the corresponding control (in the absence of propofol). Mean of data from four strips, with SD shown by vertical lines. *Significant difference from the corresponding control (P < 0.05).
Figure 7. Concentration-dependent effects of propofol on increases in [Ca (2+)]iand force induced by norepinephrine in ryanodine-treated smooth muscle strips. Actual traces of simultaneous measurements of increases in [Ca (2+)]i(Aa) and force (Ba) induced by 3 [micro sign]M norepinephrine (both sets of traces were obtained from a single smooth muscle strip). Norepinephrine was applied for 2 min at 20-min intervals in the presence or absence of propofol. Propofol was applied for 5 min before and was present during the subsequent application of norepinephrine. Right-hand panels show concentration-dependent effects of propofol on increases in [Ca2+]i(Ab) and force (Bb) induced by norepinephrine. The delta [Ca2+]ivalues (see legend to Figure 6) induced by 3 [micro sign]M norepinephrine were calculated in the presence and absence of propofol, and then the percentage inhibition induced by propofol was calculated (Ab). In (Bb), propofol-induced changes in the maximum force induced by 3 [micro sign]M norepinephrine are expressed as percentages of the corresponding control (in the absence of propofol). Mean of data from four strips, with SD shown by vertical lines. *Significant difference from the corresponding control (P < 0.05).
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Figure 8. Effect of propofol on increases in [Ca2+]iand force induced by high K+(A) or norepinephrine (B) in the presence of nicardipine in a ryanodine-treated smooth muscle strip. After the muscle strip had been treated with ryanodine (as described for Figure 5), high K (+)(128 mM, A) and norepinephrine (3 [micro sign]M, B) were applied successively for 2-min periods with a 10-min interval (left panels). This protocol was repeated after a 20-min intermission. Nicardipine (0.3 [micro sign]M) was then pretreated for 5 min and it was present throughout the rest of the experiment. High K+and norepinephrine were again successively applied for 2 min in the presence of nicardipine (middle panels), followed by a washout for 20 min. Propofol (100 [micro sign]M) was pretreated for 2 min and it was present during the subsequent application of either high K+or norepinephrine (right panels). The results illustrated were obtained from a single smooth muscle strip and were reproducible in another three strips.
Figure 8. Effect of propofol on increases in [Ca2+]iand force induced by high K+(A) or norepinephrine (B) in the presence of nicardipine in a ryanodine-treated smooth muscle strip. After the muscle strip had been treated with ryanodine (as described for Figure 5), high K (+)(128 mM, A) and norepinephrine (3 [micro sign]M, B) were applied successively for 2-min periods with a 10-min interval (left panels). This protocol was repeated after a 20-min intermission. Nicardipine (0.3 [micro sign]M) was then pretreated for 5 min and it was present throughout the rest of the experiment. High K+and norepinephrine were again successively applied for 2 min in the presence of nicardipine (middle panels), followed by a washout for 20 min. Propofol (100 [micro sign]M) was pretreated for 2 min and it was present during the subsequent application of either high K+or norepinephrine (right panels). The results illustrated were obtained from a single smooth muscle strip and were reproducible in another three strips.
Figure 8. Effect of propofol on increases in [Ca2+]iand force induced by high K+(A) or norepinephrine (B) in the presence of nicardipine in a ryanodine-treated smooth muscle strip. After the muscle strip had been treated with ryanodine (as described for Figure 5), high K (+)(128 mM, A) and norepinephrine (3 [micro sign]M, B) were applied successively for 2-min periods with a 10-min interval (left panels). This protocol was repeated after a 20-min intermission. Nicardipine (0.3 [micro sign]M) was then pretreated for 5 min and it was present throughout the rest of the experiment. High K+and norepinephrine were again successively applied for 2 min in the presence of nicardipine (middle panels), followed by a washout for 20 min. Propofol (100 [micro sign]M) was pretreated for 2 min and it was present during the subsequent application of either high K+or norepinephrine (right panels). The results illustrated were obtained from a single smooth muscle strip and were reproducible in another three strips.
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Table 1. Effects of Ryanodine on Increases in [Ca2+]iand Force Induced by 128 mM K+or 3 [micro sign]M Norepinephrine (NE) in Smooth Muscle Strips of the Rabbit Mesenteric Artery 
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Table 1. Effects of Ryanodine on Increases in [Ca2+]iand Force Induced by 128 mM K+or 3 [micro sign]M Norepinephrine (NE) in Smooth Muscle Strips of the Rabbit Mesenteric Artery 
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