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Perioperative Medicine  |   April 2016
Propofol-induced Inhibition of Catecholamine Release Is Reversed by Maintaining Calcium Influx
Author Notes
  • From the Department of Anesthesiology (L.H., X.H.) and Department of Thoracic Surgery, Dalian Municipal Friendship Hospital (L.Z.), Dalian Medical University, Dalian, Liaoning, China; Department of Pathology and Anatomy, Eastern Virginia Medical School, Norfolk, Virginia (S.F., G.L.); Department of Urology, the First Affiliated Hospital (Q.L.) and Department of Physiology (A.L.), Dalian Medical University, Dalian, Liaoning, China; Division of Fundamental Neurobiology, Toronto Western Research Institute, University Health Network, Toronto, Ontario, Canada (L.H., S.G., R.S., S.S.); and Department of Physiology, University of Toronto, Toronto, Ontario, Canada (S.S.).
  • Submitted for publication December 16, 2014. Accepted for publication December 16, 2015.
    Submitted for publication December 16, 2014. Accepted for publication December 16, 2015.×
  • Address correspondence to Dr. Han: Dalian Municipal Friendship Hospital, Dalian Medical University, 8 Sanba Square, Zhongshan District, Dalian, Liaoning 116001, China. han651310@163.com. Information on purchasing reprints may be found at www.anesthesiology.org or on the masthead page at the beginning of this issue. Anesthesiology’s articles are made freely accessible to all readers, for personal use only, 6 months from the cover date of the issue.
Article Information
Perioperative Medicine / Basic Science / Pharmacology
Perioperative Medicine   |   April 2016
Propofol-induced Inhibition of Catecholamine Release Is Reversed by Maintaining Calcium Influx
Anesthesiology 4 2016, Vol.124, 878-884. doi:10.1097/ALN.0000000000001015
Anesthesiology 4 2016, Vol.124, 878-884. doi:10.1097/ALN.0000000000001015
Abstract

Background: Propofol (2,6-diisopropylphenol) is one of the most frequently used anesthetic agents. One of the main side effects of propofol is to reduce blood pressure, which is thought to occur by inhibiting the release of catecholamines from sympathetic neurons. Here, the authors hypothesized that propofol-induced hypotension is not simply the result of suppression of the release mechanisms for catecholamines.

Methods: The authors simultaneously compared the effects of propofol on the release of norepinephrine triggered by high K+-induced depolarization, as well as ionomycin, by using neuroendocrine PC12 cells and synaptosomes. Ionomycin, a Ca2+ ionophore, directly induces Ca2+ influx, thus bypassing the effect of ion channel modulation by propofol.

Results: Propofol decreased depolarization (high K+)-triggered norepinephrine release, whereas it increased ionomycin-triggered release from both PC12 cells and synaptosomes. The propofol (30 μM)-induced increase in norepinephrine release triggered by ionomycin was dependent on both the presence and the concentration of extracellular Ca2+ (0.3 to 10 mM; n = 6). The enhancement of norepinephrine release by propofol was observed in all tested concentrations of ionomycin (0.1 to 5 μM; n = 6).

Conclusions: Propofol at clinically relevant concentrations promotes the catecholamine release as long as Ca2+ influx is supported. This unexpected finding will allow for a better understanding in preventing propofol-induced hypotension.

Abstract

Clinically relevant concentrations of propofol (10 to 30 μM) indeed inhibited the depolarization (by K+)-dependent norepinephrine release in cultured PC12 cells (which are derived from rat adrenal chromaffin cells) and synaptosomes. Unexpectedly, the same propofol concentrations that inhibited depolarization-dependent norepinephrine release increased ionomycin (a Ca2+ ionophore)-triggered catecholamine release in a Ca2+-dependent manner. The Ca2+-dependent propofol-induced increase in ionomycin-triggered catecholamine release was abolished in the presence of the Ca2+ chelator, EGTA.

What We Already Know about This Topic
  • Propofol-induced hypotension is believed to be partly caused by the inhibition of catecholamine release in the sympathetic nervous system

What This Article Tells Us That Is New
  • Clinically relevant concentrations of propofol (10 to 30 μM) indeed inhibited the depolarization (by K+)-dependent norepinephrine release in cultured PC12 cells (which are derived from rat adrenal chromaffin cells) and synaptosomes

  • Unexpectedly, the same propofol concentrations that inhibited depolarization-dependent norepinephrine release increased ionomycin (a Ca2+ ionophore)-triggered catecholamine release in a Ca2+-dependent manner

  • The Ca2+-dependent propofol-induced increase in ionomycin-triggered catecholamine release was abolished in the presence of the Ca2+ chelator, EGTA

PROPOFOL (2,6-diisopropylphenol) is a widely used intravenous anesthetic for induction and maintenance of general anesthesia or sedation. However, the use of propofol is often accompanied by a significant reduction in arterial blood pressure.1,2  Various mechanisms associated with propofol-induced hypotension have been reported. Examples include direct myocardial depression by inhibition of Ca2+-induced excitation–contraction coupling, as well as reduced Ca2+ influx,3–6  endothelial-dependent and endothelial-independent inhibition of vascular tone via opening of potassium channels to produce hyperpolarization and reduction of Ca2+-induced force generation.7–11  In addition, the key inhibitory effect of propofol on the cardiovascular system is reported to be due to an inhibition of the sympathetic nervous system,12–16  which results in decreased catecholamine release.17  For example, propofol at clinically relevant concentrations (2.5 mg/kg body weight) significantly decreased arterial blood pressure and serum norepinephrine levels in rats. In addition, propofol (10 to 50 μM) inhibited catecholamine secretion in a dose-dependent manner by carbachol stimulation in cultured adrenal medullary cells. However, it remains to be determined whether this inhibition is due to the modulation of ion channels that facilitate Ca2+ influx17  or is due to the inhibition of the release machinery itself. Recent reports have shown that propofol inhibits the catecholamine release machinery in PC12 cell lines and adrenal chromaffin cells by interacting with Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) proteins and/or SNARE-associated proteins, such as syntaxin1, synaptotagmin I, synaptosomal-associated protein, 25kDa) (SNAP-25), and SNAP-23.18,19  The evidence was obtained from amperometric recordings of permeabilized cells that were directly stimulated by Ca2+. However, these results need to be confirmed using intact cells. We hypothesize that, contrary to the current belief, propofol does not inhibit the machinery mediating catecholamine release but rather enhances it. To test this hypothesis, we simultaneously compared the effects of propofol on the release of norepinephrine triggered by depolarization (70 mM, KCl) and ionomycin (1 μM) using neuroendocrine PC12 cells and synaptosomes.20,21  Ionomycin, a Ca2+ ionophore, can directly induce Ca2+ influx, thus bypassing the effect of ion channel modulation by propofol.22 
Materials and Methods
General Materials
Propofol (2,6-diisopropylphenol, D126608) was purchased from Sigma Chemical, Canada. Ionomycin was purchased from Cayman Chemicals (no. 10004974, USA). [3H] norepinephrine (NET377) was purchased from PerkinElmer (USA).
PC12 Cell Culture
PC12 cells are derived from rat adrenal chromaffin cells and have the ability to differentiate and exhibit the phenotypes of sympathetic neurons when exposed to nerve growth factor, thus making these cells ideal models for both chromaffin cells and sympathetic neurons.20  Dopamine and norepinephrine are the major catecholamines in PC12 cells,20  whereas norepinephrine and epinephrine are the major catecholamines in adrenal chromaffin cells. The culture of PC12 cells was previously described.23–27  Briefly, PC12 cells were routinely cultured in Dulbecco’s modified Eagle’s medium, containing 4.5 mM glucose, 5% calf serum (iron supplemented), 5% equine serum, 100 U/ml penicillin–streptomycin, and 0.25 μg/ml amphotericin B. The cells were incubated at 37°C, 7.5% CO2, and were passaged once a week.
[3H] Norepinephrine Release Assays Using Intact PC12 Cells
PC12 cells were plated in 24-well plates; 3 to 4 days after plating, the cells were labeled with 0.5 μCi of [3H] norepinephrine in the presence of 0.5 mM of ascorbic acid for 12 to 16 h. The labeled PC12 cells were incubated with fresh complete Dulbecco’s modified Eagle’s medium for 1 to 3 h to remove unincorporated [3H] norepinephrine. The cells were washed twice with physiological saline solution (PSS) containing 145 mM NaCl, 5.6 mM KCl, 2.2 mM CaCl2, 0.5 mM MgCl2, 5.6 mM glucose, and 15 mM HEPES, pH 7.4. The norepinephrine secretion was triggered with 200 μl of PSS, high K+-PSS (containing 81 mM NaCl and 70 mM KCl), or the indicated concentration of ionomycin in the presence or absence of propofol. Different concentrations of Ca2+ were prepared with the same formula as PSS. Different concentrations of propofol were made and mixed with PSS, high K+-PSS, or ionomycin-PSS. Secretion was terminated after a 10-min incubation at 37°C by chilling to 0°C, and samples were centrifuged at 4°C for 3 min. Supernatants were removed, and the pellets were solubilized in 0.1% Triton X-100 for liquid scintillation counting. We chose six as the number of samples (n = 6) for PC12 release assays based on our previous experience showing that this number is usually sufficient to show the significance. We did not use the randomization or the blinding methods.
[3H] Norepinephrine Release Assays Using Synaptosomes
Synaptosomes were prepared as described,28  using rat cortical regions, and resuspended in 10 volume of aerated (95% O2, 5% CO2) Krebs-bicarbonate buffer (pH 7.4; composition in mM: NaCl 118, KCl 3.5, CaCl2 1.25, MgSO4 1.2, KH2PO4, 1.2, NaHCO3 25, and D-glucose 11.5) and placed on ice.28–30  After 90 min of equilibration on ice, synaptosomes were incubated with 0.2 μM [3H] norepinephrine for 5 min at 35°C. The synaptosomes were trapped on a glass fiber filter and superfused at 33°C with Krebs-bicarbonate buffer with a flow rate of 0.8 ml/min under continuous aeration with 95% O2/5% CO2. After 12 min of preincubation (superfusion) with propofol (100 μM) or control dimethyl sulfoxide (DMSO), two 1-min fractions were collected to determine the basal norepinephrine release. A 30-s pulse stimulus (Krebs-bicarbonate buffer containing 5 μM ionomycin or 25 mM KCl) was then applied to trigger release. The superfusate was collected continuously, while the [3H] norepinephrine content of the superfusate and the synaptosomes at the end of superfusion was determined by liquid scintillation counting. Release of [3H] norepinephrine, expressed as the fractional release rate, was calculated as the fraction of norepinephrine radioactivity released, divided by the amount remaining on the filter at that particular point in time. We combined the data from the same animal, and the release in the control condition was normalized and set to 100%.
Statistical Analysis
To examine the significance of the dose-dependent effects of propofol on norepinephrine release in PSS, high K+, or ionomycin, we performed one-way ANOVA with repeated measures followed by post hoc Bonferroni test using the js-STAR 2012 program, which is freely available online (http://www.kisnet.or.jp/nappa/software/star/index.htm; fig. 1).
Fig. 1.
Propofol (10, 30, and 100 μM) inhibits the norepinephrine release triggered by high K+ and facilitates the norepinephrine release by ionomycin in a concentration-dependent manner. Propofol: dissolved in dimethyl sulfoxide (DMSO), the final concentration of DMSO is lower than 0.1%. Error bars indicate SD (n = 6). One-way ANOVA with repeated measures revealed significant, dose-dependent effects of propofol on norepinephrine release in physiological saline solution (PSS; P < 0.0001; 100 μM propofol stimulated significantly higher release than others based on post hoc Bonferroni test, P < 0.05), high K+ (P < 0.0001; post hoc test showed significant differences in all the paired comparisons, P < 0.05), or ionomycin (P < 0.0001; post hoc test showed significant differences in all the paired comparisons except for DMSO versus 10 μM propofol, P < 0.05). *P < 0.05, and n.s. indicates not significant at P = 0.05 level. High K+ = 70 mM potassium chloride (KCl); ionomycin = 1 μM.
Propofol (10, 30, and 100 μM) inhibits the norepinephrine release triggered by high K+ and facilitates the norepinephrine release by ionomycin in a concentration-dependent manner. Propofol: dissolved in dimethyl sulfoxide (DMSO), the final concentration of DMSO is lower than 0.1%. Error bars indicate SD (n = 6). One-way ANOVA with repeated measures revealed significant, dose-dependent effects of propofol on norepinephrine release in physiological saline solution (PSS; P < 0.0001; 100 μM propofol stimulated significantly higher release than others based on post hoc Bonferroni test, P < 0.05), high K+ (P < 0.0001; post hoc test showed significant differences in all the paired comparisons, P < 0.05), or ionomycin (P < 0.0001; post hoc test showed significant differences in all the paired comparisons except for DMSO versus 10 μM propofol, P < 0.05). *P < 0.05, and n.s. indicates not significant at P = 0.05 level. High K+ = 70 mM potassium chloride (KCl); ionomycin = 1 μM.
Fig. 1.
Propofol (10, 30, and 100 μM) inhibits the norepinephrine release triggered by high K+ and facilitates the norepinephrine release by ionomycin in a concentration-dependent manner. Propofol: dissolved in dimethyl sulfoxide (DMSO), the final concentration of DMSO is lower than 0.1%. Error bars indicate SD (n = 6). One-way ANOVA with repeated measures revealed significant, dose-dependent effects of propofol on norepinephrine release in physiological saline solution (PSS; P < 0.0001; 100 μM propofol stimulated significantly higher release than others based on post hoc Bonferroni test, P < 0.05), high K+ (P < 0.0001; post hoc test showed significant differences in all the paired comparisons, P < 0.05), or ionomycin (P < 0.0001; post hoc test showed significant differences in all the paired comparisons except for DMSO versus 10 μM propofol, P < 0.05). *P < 0.05, and n.s. indicates not significant at P = 0.05 level. High K+ = 70 mM potassium chloride (KCl); ionomycin = 1 μM.
×
To examine the significance of propofol’s effects on ionomycin-induced and depolarization-induced norepinephrine release from synaptosomes, we performed the paired t test: two samples assuming unequal variances using Excel (Microsoft, USA). The n indicates the number of animals used. We combined data from the same animal (fig. 2).
Fig. 2.
Propofol enhances ionomycin-induced norepinephrine (NE) release (A), while it inhibits norepinephrine release triggered by high K+ (B). Synaptosomes from rat cortical regions were preincubated with propofol (100 μM) or control dimethyl sulfoxide and stimulated for 30 s with 5 μM ionomycin or 25 mM potassium chloride (KCl). Error bars indicate SD (n = 5 for A, n = 3 for B). Paired t test showed that a 12-min preincubation with 100 μM propofol significantly enhanced the ionomycin (5 μM)-triggered release (paired t test, t4 = 3.21, P = 0.016), while it inhibited the depolarization (25 mM KCl)-triggered release (t2 = 4.66, P = 0.02). *P < 0.05.
Propofol enhances ionomycin-induced norepinephrine (NE) release (A), while it inhibits norepinephrine release triggered by high K+ (B). Synaptosomes from rat cortical regions were preincubated with propofol (100 μM) or control dimethyl sulfoxide and stimulated for 30 s with 5 μM ionomycin or 25 mM potassium chloride (KCl). Error bars indicate SD (n = 5 for A, n = 3 for B). Paired t test showed that a 12-min preincubation with 100 μM propofol significantly enhanced the ionomycin (5 μM)-triggered release (paired t test, t4 = 3.21, P = 0.016), while it inhibited the depolarization (25 mM KCl)-triggered release (t2 = 4.66, P = 0.02). *P < 0.05.
Fig. 2.
Propofol enhances ionomycin-induced norepinephrine (NE) release (A), while it inhibits norepinephrine release triggered by high K+ (B). Synaptosomes from rat cortical regions were preincubated with propofol (100 μM) or control dimethyl sulfoxide and stimulated for 30 s with 5 μM ionomycin or 25 mM potassium chloride (KCl). Error bars indicate SD (n = 5 for A, n = 3 for B). Paired t test showed that a 12-min preincubation with 100 μM propofol significantly enhanced the ionomycin (5 μM)-triggered release (paired t test, t4 = 3.21, P = 0.016), while it inhibited the depolarization (25 mM KCl)-triggered release (t2 = 4.66, P = 0.02). *P < 0.05.
×
To examine the significance of propofol’s effects on ionomycin-induced norepinephrine release, we performed two-way ANOVA with repeated measures followed by post hoc Bonferroni test using the js-STAR 2012 program (fig. 3).
Fig. 3.
Propofol promotes the norepinephrine release triggered by different concentrations (0.3 to 10 μM) of ionomycin. −Propofol: control cell group incubated with dimethyl sulfoxide (less than 0.1%). +Propofol: experimental cell group incubated with 30 μM propofol. Ionomycin: 0, 0.1, 0.3, 1, 2.5, and 5 μM. Note that without ionomycin, the norepinephrine secretion level is similar with or without propofol incubation. Error bars indicate SD (n = 6). Two-way ANOVA revealed significant effects due to both propofol application (P < 0.0005) and the dose of ionomycin used (P < 0.0001). There was a significant interaction between the presence of propofol and the concentration of ionomycin (P < 0.0001). *Significant (P < 0.05) differences based on post hoc Bonferroni test.
Propofol promotes the norepinephrine release triggered by different concentrations (0.3 to 10 μM) of ionomycin. −Propofol: control cell group incubated with dimethyl sulfoxide (less than 0.1%). +Propofol: experimental cell group incubated with 30 μM propofol. Ionomycin: 0, 0.1, 0.3, 1, 2.5, and 5 μM. Note that without ionomycin, the norepinephrine secretion level is similar with or without propofol incubation. Error bars indicate SD (n = 6). Two-way ANOVA revealed significant effects due to both propofol application (P < 0.0005) and the dose of ionomycin used (P < 0.0001). There was a significant interaction between the presence of propofol and the concentration of ionomycin (P < 0.0001). *Significant (P < 0.05) differences based on post hoc Bonferroni test.
Fig. 3.
Propofol promotes the norepinephrine release triggered by different concentrations (0.3 to 10 μM) of ionomycin. −Propofol: control cell group incubated with dimethyl sulfoxide (less than 0.1%). +Propofol: experimental cell group incubated with 30 μM propofol. Ionomycin: 0, 0.1, 0.3, 1, 2.5, and 5 μM. Note that without ionomycin, the norepinephrine secretion level is similar with or without propofol incubation. Error bars indicate SD (n = 6). Two-way ANOVA revealed significant effects due to both propofol application (P < 0.0005) and the dose of ionomycin used (P < 0.0001). There was a significant interaction between the presence of propofol and the concentration of ionomycin (P < 0.0001). *Significant (P < 0.05) differences based on post hoc Bonferroni test.
×
To examine the significance of propofol’s effects and the different concentrations of extracellular Ca2+ on ionomycin-induced norepinephrine release, we performed two-way ANOVA with repeated measures followed by post hoc Bonferroni test using the js-STAR 2012 program (fig. 4).
Fig. 4.
Propofol increases norepinephrine secretion in a Ca2+-dependent manner. Ionomycin: 1 μM. −Propofol: control cell group incubated with dimethyl sulfoxide (less than 0.1%). +Propofol: experimental cell group incubated with 30 μM propofol. Extracellular Ca2+ concentration: 0 (= 0.1 mM EGTA), 0.3 mM, 1 mM, 2.2 mM, 5 mM, and 10 mM. Note that without extracellular Ca2+, the norepinephrine secretion level is similar with or without propofol incubation. Error bars indicate SD (n = 6). Two-way ANOVA revealed significant effects of both propofol (P = 0.0003) and the concentration of extracellular Ca2+ (P < 0.0001). There was also a significant interaction between the presence of propofol and the concentration of extracellular Ca2+ (P < 0.0001). *Significant (P < 0.05) differences based on post hoc Bonferroni test. n.s. indicates not significant at P = 0.05 level.
Propofol increases norepinephrine secretion in a Ca2+-dependent manner. Ionomycin: 1 μM. −Propofol: control cell group incubated with dimethyl sulfoxide (less than 0.1%). +Propofol: experimental cell group incubated with 30 μM propofol. Extracellular Ca2+ concentration: 0 (= 0.1 mM EGTA), 0.3 mM, 1 mM, 2.2 mM, 5 mM, and 10 mM. Note that without extracellular Ca2+, the norepinephrine secretion level is similar with or without propofol incubation. Error bars indicate SD (n = 6). Two-way ANOVA revealed significant effects of both propofol (P = 0.0003) and the concentration of extracellular Ca2+ (P < 0.0001). There was also a significant interaction between the presence of propofol and the concentration of extracellular Ca2+ (P < 0.0001). *Significant (P < 0.05) differences based on post hoc Bonferroni test. n.s. indicates not significant at P = 0.05 level.
Fig. 4.
Propofol increases norepinephrine secretion in a Ca2+-dependent manner. Ionomycin: 1 μM. −Propofol: control cell group incubated with dimethyl sulfoxide (less than 0.1%). +Propofol: experimental cell group incubated with 30 μM propofol. Extracellular Ca2+ concentration: 0 (= 0.1 mM EGTA), 0.3 mM, 1 mM, 2.2 mM, 5 mM, and 10 mM. Note that without extracellular Ca2+, the norepinephrine secretion level is similar with or without propofol incubation. Error bars indicate SD (n = 6). Two-way ANOVA revealed significant effects of both propofol (P = 0.0003) and the concentration of extracellular Ca2+ (P < 0.0001). There was also a significant interaction between the presence of propofol and the concentration of extracellular Ca2+ (P < 0.0001). *Significant (P < 0.05) differences based on post hoc Bonferroni test. n.s. indicates not significant at P = 0.05 level.
×
Results
Propofol Decreased High K+-triggered Norepinephrine Release, Whereas It Increased Ionomycin-triggered Release in a Dose-dependent Manner
To determine the exact site and mechanism through which propofol inhibits catecholamine release, we measured the effects of an acute application of propofol (10, 30, and 100 μM) on [3H] norepinephrine release from intact PC12 cells. [3H] norepinephrine release was triggered by high K+-induced depolarization (70 mM KCl, n = 6) or by the application of ionomycin (1 μM, n = 6).13–17  In the control group (i.e., no propofol, n = 6), cells were incubated with less than 0.1% DMSO, which was used to dissolve the propofol. We found that propofol inhibited high K+-triggered [3H] norepinephrine release in a dose-dependent manner (one-way ANOVA with repeated measures, P < 0.0001; post hoc Bonferroni test shows significant differences in all the paired comparisons, P < 0.05). In the control group, high K+-induced depolarization triggered [3H] norepinephrine release by approximately six times that of the PSS, whereas in the presence of 30 μM propofol, high K+ triggered [3H] norepinephrine release by approximately 3 times that of the PSS. Furthermore, 100 μM propofol almost completely inhibited the stimulation by high K+. Thus, propofol significantly inhibited depolarization-triggered catecholamine release, which is consistent with the findings of Yamazaki et al.8  Propofol concentrations of 10 and 30 μM had no effect on the baseline secretion of norepinephrine in the PSS, whereas 100 μM propofol weakly stimulated it (fig. 1) (P < 0.0001; 100 μM propofol stimulated significantly higher release than others based on post hoc test, P < 0.05).
In contrast, we unexpectedly found that propofol enhanced ionomycin-triggered [3H] norepinephrine release, which occurred in a dose-dependent manner (fig. 1; P < 0.0001; post hoc test shows significant differences in all the paired comparisons except for DMSO vs. 10 μM propofol, P < 0.05). Thirty micromolar propofol increased [3H] norepinephrine release by approximately 40% compared with the control DMSO group, while 100 μM propofol increased release by more than 100% (fig. 1). This unexpected result suggests that propofol enhances the release machinery of catecholamines in PC12 cells. Next, we tested whether our finding was also applicable to norepinephrine release from primary neurons. For this purpose, we performed [3H] norepinephrine release assays using rat cortical synaptosomes. We found that a 12-min incubation with 100 μM propofol enhances the ionomycin (5 μM)-triggered release (paired t test, t4 = 3.21, P = 0.016). However, it inhibits the depolarization (25 mM KCl)-triggered release (t2 = 4.66, P = 0.02). Thus, the results observed in PC12 cells are consistent with those observed in rat synaptosomes.
To examine whether the stimulatory effect of propofol on ionomycin-triggered catecholamine release can be consistently observed, we tested different concentrations of ionomycin (0, 0.1, 0.3, 1, 2.5, and 5 μM) (fig. 3). In the DMSO control group (n = 6), ionomycin increased [3H] norepinephrine release in a dose-dependent manner. An important finding was that 30 μM propofol (n = 6) consistently enhanced the ionomycin-triggered [3H] norepinephrine release in all the ionomycin concentrations tested (fig. 3). Two-way ANOVA with repeated measures revealed significant effects due to both propofol application (P < 0.0005) and the dose of ionomycin used (P < 0.0001). The enhancement of norepinephrine secretion levels by propofol is clearly dependent on the presence of ionomycin because in the absence of ionomycin, norepinephrine secretion levels are similar with or without propofol incubation. There was a significant interaction between the presence of propofol and the concentration of ionomycin (P < 0.0001). Thus, propofol consistently stimulated ionomycin-triggered [3H] norepinephrine release in an ionomycin-dependent manner.
Propofol Enhances Ionomycin-mediated Norepinephrine Secretion in a Ca2+-dependent Manner
The next important question is: Does propofol (30 μM) promote the catecholamine release machinery in a Ca2+-dependent manner? To address this question, we titrated various concentrations of extracellular Ca2+ (0, 0.3, 1, 2.2, 5, and 10 mM) and triggered secretion with a fixed concentration of ionomycin (1 μM; fig. 4). We hypothesized that if propofol does promote Ca2+-dependent catecholamine release, the stimulatory effects of propofol should disappear in the absence of Ca2+ (with 0.1 mM EGTA). We indeed found that there was no difference in [3H] norepinephrine release between the propofol group (n = 6) and the control DMSO group (n = 6) in the absence of Ca2+. However, in the presence of extracellular Ca2+ (0.3, 1, 2.2, 5, and 10 mM), propofol did enhance [3H] norepinephrine release. Two-way ANOVA revealed significant effects of both propofol (P = 0.0003) and the concentration of extracellular Ca2+ (P < 0.0001). There was also a significant interaction between the presence of propofol and the concentration of extracellular Ca2+ (P < 0.0001). The stimulatory effect seemed particularly evident at extracellular Ca2+ concentrations of 2.2 and 5 mM. Thus, propofol (30 μM) specifically promotes Ca2+-dependent, regulated catecholamine secretion, while it has no effect on constitutive catecholamine secretion in the absence of Ca2+. Therefore, we conclude that the inhibition of depolarization-triggered catecholamine release by propofol (fig. 1) is not due to inhibition of the release machinery itself.
Discussion
Propofol is a sedative and an anesthetic drug used to induce and maintain general anesthesia in the operating room or intensive care unit. In the clinical setting, the recommended dose of propofol for induction is 1.5 to 2.5 mg/kg for adults. Anesthesia occurs at blood concentrations of 4 to 6 mg/l, which corresponds with 22 to 34 μM. These doses of propofol may lead to a significant reduction in arterial blood pressure in patients.1,2  This propofol-induced hypotension is believed to be partly caused by the inhibition of catecholamine release in the sympathetic nervous system.12–16  Indeed, our results confirm that clinically relevant concentrations of propofol (10 to 30 μM) inhibit the depolarization-dependent norepinephrine secretion in cultured PC12 cells and synaptosomes (figs. 1 and 2). Unexpectedly however, we also found that the same concentrations of propofol increase the ionomycin-triggered catecholamine release in a Ca2+-dependent manner (figs. 1–4). Ca2+ dependence was clearly demonstrated by the abolished stimulation by propofol in the presence of the Ca2+ chelator, EGTA (0.1 mM) (fig. 4). Furthermore, potentiation of the release was consistently observed over a wide range of ionomycin concentrations that were tested (fig. 3). Therefore, our results indicate that the inhibition of depolarization (by high-K+)-triggered catecholamine release by propofol (figs. 1 and 2) is likely due to modulation of the ion channels and impairment of the Ca2+ influx, without any interference of the neurotransmitter release machinery itself.
We found that the enhancing effect of propofol on ionomycin-induced Ca2+-dependent norepinephrine secretion is observed in the wide range of concentrations of ionomycin and extracellular Ca2+ that were tested (figs. 3 and 4). Although we did not directly measure and compare the intracellular concentration of Ca2+ in response to depolarization versus ionomycin, our results in figures 1, 3, and 4 indicate that the Ca2+ increase induced by ionomycin is similar to the Ca2+ increase induced by depolarization for the reasons discussed later. In figure 4, we examined the effects of the titrated concentrations of extracellular Ca2+ on norepinephrine release and found that the higher the extracellular Ca2+, the higher the norepinephrine release. This suggests that the influx of intracellular Ca2+ caused by ionomycin (1 μM) is not saturated in the range of extracellular Ca2+ we tested (0.3, 1, 2.2, 5, and 10 mM). Similarly, in figure 3, we observed that the higher the ionomycin concentration, the higher the norepinephrine release, suggesting that the influx of intracellular Ca2+ caused by different concentrations of ionomycin (0.1, 0.3, 1, 2.5, and 5 μM) is not saturated in 2.2 mM extracellular Ca2+. The release level triggered by 1 μM ionomycin with 5 mM extracellular Ca2+ (fig. 4) and the release by 5 μM ionomycin with 2.2 mM extracellular Ca2+ (fig. 3) are comparable with the release triggered by high K+ (70 mM) with 2.2 mM extracellular Ca2+ (approximately 25% of norepinephrine release in fig. 1). Thus, the ionomycin-induced Ca2+ influx in this study seems comparable with the depolarization-induced Ca2+ influx. Thus, our results strongly suggest that the facilitating effect of propofol is observed in the physiological range of increases in intracellular Ca2+ caused by depolarization. The mechanism by which propofol facilitates Ca2+-dependent release will be an interesting topic for future study.
Our results seem to agree with the vast array of literature, suggesting that propofol causes an inhibition of Ca2+ current and an increase in various K+ currents in vascular and cardiac cells.3–12  Similar modulations of ionic currents in PC12 cells and sympathetic neurons would lead to a decrease in depolarization-induced Ca2+ influx, which results in the inhibition of catecholamine release. An unexpected finding of the present study is the discovery of the enhancement of the machinery that mediates catecholamine release. Our work does not agree with recent studies which suggest that propofol decreases catecholamine release by inhibiting the release machinery itself via interactions with SNARE proteins or SNARE-interacting proteins.18,19 
In summary, contrary to the current belief, clinically relevant propofol concentrations enhance the release machinery of catecholamines if Ca2+ influx is maintained. The data in the present study suggest that modulation of ion channels rather than alteration of vesicle release mechanisms contributes to propofol-induced reductions in synaptic catecholamine concentration. This appears to be an additional pathway along with other known effects on vascular smooth muscle and myocardial function that may contribute to propofol-induced hemodynamic changes in the clinical setting.
Acknowledgments
Supported by grant nos. 81150022 and 81350015 from National Natural Science Foundation of China, Beijing, China (to Dr. Han) and by operating grant (MOP-130573) from Canadian Institutes of Health Research, Ottawa, Ontario, Canada (to Dr. Sugita).
Competing Interests
The authors declare no competing interests.
References
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Fig. 1.
Propofol (10, 30, and 100 μM) inhibits the norepinephrine release triggered by high K+ and facilitates the norepinephrine release by ionomycin in a concentration-dependent manner. Propofol: dissolved in dimethyl sulfoxide (DMSO), the final concentration of DMSO is lower than 0.1%. Error bars indicate SD (n = 6). One-way ANOVA with repeated measures revealed significant, dose-dependent effects of propofol on norepinephrine release in physiological saline solution (PSS; P < 0.0001; 100 μM propofol stimulated significantly higher release than others based on post hoc Bonferroni test, P < 0.05), high K+ (P < 0.0001; post hoc test showed significant differences in all the paired comparisons, P < 0.05), or ionomycin (P < 0.0001; post hoc test showed significant differences in all the paired comparisons except for DMSO versus 10 μM propofol, P < 0.05). *P < 0.05, and n.s. indicates not significant at P = 0.05 level. High K+ = 70 mM potassium chloride (KCl); ionomycin = 1 μM.
Propofol (10, 30, and 100 μM) inhibits the norepinephrine release triggered by high K+ and facilitates the norepinephrine release by ionomycin in a concentration-dependent manner. Propofol: dissolved in dimethyl sulfoxide (DMSO), the final concentration of DMSO is lower than 0.1%. Error bars indicate SD (n = 6). One-way ANOVA with repeated measures revealed significant, dose-dependent effects of propofol on norepinephrine release in physiological saline solution (PSS; P < 0.0001; 100 μM propofol stimulated significantly higher release than others based on post hoc Bonferroni test, P < 0.05), high K+ (P < 0.0001; post hoc test showed significant differences in all the paired comparisons, P < 0.05), or ionomycin (P < 0.0001; post hoc test showed significant differences in all the paired comparisons except for DMSO versus 10 μM propofol, P < 0.05). *P < 0.05, and n.s. indicates not significant at P = 0.05 level. High K+ = 70 mM potassium chloride (KCl); ionomycin = 1 μM.
Fig. 1.
Propofol (10, 30, and 100 μM) inhibits the norepinephrine release triggered by high K+ and facilitates the norepinephrine release by ionomycin in a concentration-dependent manner. Propofol: dissolved in dimethyl sulfoxide (DMSO), the final concentration of DMSO is lower than 0.1%. Error bars indicate SD (n = 6). One-way ANOVA with repeated measures revealed significant, dose-dependent effects of propofol on norepinephrine release in physiological saline solution (PSS; P < 0.0001; 100 μM propofol stimulated significantly higher release than others based on post hoc Bonferroni test, P < 0.05), high K+ (P < 0.0001; post hoc test showed significant differences in all the paired comparisons, P < 0.05), or ionomycin (P < 0.0001; post hoc test showed significant differences in all the paired comparisons except for DMSO versus 10 μM propofol, P < 0.05). *P < 0.05, and n.s. indicates not significant at P = 0.05 level. High K+ = 70 mM potassium chloride (KCl); ionomycin = 1 μM.
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Fig. 2.
Propofol enhances ionomycin-induced norepinephrine (NE) release (A), while it inhibits norepinephrine release triggered by high K+ (B). Synaptosomes from rat cortical regions were preincubated with propofol (100 μM) or control dimethyl sulfoxide and stimulated for 30 s with 5 μM ionomycin or 25 mM potassium chloride (KCl). Error bars indicate SD (n = 5 for A, n = 3 for B). Paired t test showed that a 12-min preincubation with 100 μM propofol significantly enhanced the ionomycin (5 μM)-triggered release (paired t test, t4 = 3.21, P = 0.016), while it inhibited the depolarization (25 mM KCl)-triggered release (t2 = 4.66, P = 0.02). *P < 0.05.
Propofol enhances ionomycin-induced norepinephrine (NE) release (A), while it inhibits norepinephrine release triggered by high K+ (B). Synaptosomes from rat cortical regions were preincubated with propofol (100 μM) or control dimethyl sulfoxide and stimulated for 30 s with 5 μM ionomycin or 25 mM potassium chloride (KCl). Error bars indicate SD (n = 5 for A, n = 3 for B). Paired t test showed that a 12-min preincubation with 100 μM propofol significantly enhanced the ionomycin (5 μM)-triggered release (paired t test, t4 = 3.21, P = 0.016), while it inhibited the depolarization (25 mM KCl)-triggered release (t2 = 4.66, P = 0.02). *P < 0.05.
Fig. 2.
Propofol enhances ionomycin-induced norepinephrine (NE) release (A), while it inhibits norepinephrine release triggered by high K+ (B). Synaptosomes from rat cortical regions were preincubated with propofol (100 μM) or control dimethyl sulfoxide and stimulated for 30 s with 5 μM ionomycin or 25 mM potassium chloride (KCl). Error bars indicate SD (n = 5 for A, n = 3 for B). Paired t test showed that a 12-min preincubation with 100 μM propofol significantly enhanced the ionomycin (5 μM)-triggered release (paired t test, t4 = 3.21, P = 0.016), while it inhibited the depolarization (25 mM KCl)-triggered release (t2 = 4.66, P = 0.02). *P < 0.05.
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Fig. 3.
Propofol promotes the norepinephrine release triggered by different concentrations (0.3 to 10 μM) of ionomycin. −Propofol: control cell group incubated with dimethyl sulfoxide (less than 0.1%). +Propofol: experimental cell group incubated with 30 μM propofol. Ionomycin: 0, 0.1, 0.3, 1, 2.5, and 5 μM. Note that without ionomycin, the norepinephrine secretion level is similar with or without propofol incubation. Error bars indicate SD (n = 6). Two-way ANOVA revealed significant effects due to both propofol application (P < 0.0005) and the dose of ionomycin used (P < 0.0001). There was a significant interaction between the presence of propofol and the concentration of ionomycin (P < 0.0001). *Significant (P < 0.05) differences based on post hoc Bonferroni test.
Propofol promotes the norepinephrine release triggered by different concentrations (0.3 to 10 μM) of ionomycin. −Propofol: control cell group incubated with dimethyl sulfoxide (less than 0.1%). +Propofol: experimental cell group incubated with 30 μM propofol. Ionomycin: 0, 0.1, 0.3, 1, 2.5, and 5 μM. Note that without ionomycin, the norepinephrine secretion level is similar with or without propofol incubation. Error bars indicate SD (n = 6). Two-way ANOVA revealed significant effects due to both propofol application (P < 0.0005) and the dose of ionomycin used (P < 0.0001). There was a significant interaction between the presence of propofol and the concentration of ionomycin (P < 0.0001). *Significant (P < 0.05) differences based on post hoc Bonferroni test.
Fig. 3.
Propofol promotes the norepinephrine release triggered by different concentrations (0.3 to 10 μM) of ionomycin. −Propofol: control cell group incubated with dimethyl sulfoxide (less than 0.1%). +Propofol: experimental cell group incubated with 30 μM propofol. Ionomycin: 0, 0.1, 0.3, 1, 2.5, and 5 μM. Note that without ionomycin, the norepinephrine secretion level is similar with or without propofol incubation. Error bars indicate SD (n = 6). Two-way ANOVA revealed significant effects due to both propofol application (P < 0.0005) and the dose of ionomycin used (P < 0.0001). There was a significant interaction between the presence of propofol and the concentration of ionomycin (P < 0.0001). *Significant (P < 0.05) differences based on post hoc Bonferroni test.
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Fig. 4.
Propofol increases norepinephrine secretion in a Ca2+-dependent manner. Ionomycin: 1 μM. −Propofol: control cell group incubated with dimethyl sulfoxide (less than 0.1%). +Propofol: experimental cell group incubated with 30 μM propofol. Extracellular Ca2+ concentration: 0 (= 0.1 mM EGTA), 0.3 mM, 1 mM, 2.2 mM, 5 mM, and 10 mM. Note that without extracellular Ca2+, the norepinephrine secretion level is similar with or without propofol incubation. Error bars indicate SD (n = 6). Two-way ANOVA revealed significant effects of both propofol (P = 0.0003) and the concentration of extracellular Ca2+ (P < 0.0001). There was also a significant interaction between the presence of propofol and the concentration of extracellular Ca2+ (P < 0.0001). *Significant (P < 0.05) differences based on post hoc Bonferroni test. n.s. indicates not significant at P = 0.05 level.
Propofol increases norepinephrine secretion in a Ca2+-dependent manner. Ionomycin: 1 μM. −Propofol: control cell group incubated with dimethyl sulfoxide (less than 0.1%). +Propofol: experimental cell group incubated with 30 μM propofol. Extracellular Ca2+ concentration: 0 (= 0.1 mM EGTA), 0.3 mM, 1 mM, 2.2 mM, 5 mM, and 10 mM. Note that without extracellular Ca2+, the norepinephrine secretion level is similar with or without propofol incubation. Error bars indicate SD (n = 6). Two-way ANOVA revealed significant effects of both propofol (P = 0.0003) and the concentration of extracellular Ca2+ (P < 0.0001). There was also a significant interaction between the presence of propofol and the concentration of extracellular Ca2+ (P < 0.0001). *Significant (P < 0.05) differences based on post hoc Bonferroni test. n.s. indicates not significant at P = 0.05 level.
Fig. 4.
Propofol increases norepinephrine secretion in a Ca2+-dependent manner. Ionomycin: 1 μM. −Propofol: control cell group incubated with dimethyl sulfoxide (less than 0.1%). +Propofol: experimental cell group incubated with 30 μM propofol. Extracellular Ca2+ concentration: 0 (= 0.1 mM EGTA), 0.3 mM, 1 mM, 2.2 mM, 5 mM, and 10 mM. Note that without extracellular Ca2+, the norepinephrine secretion level is similar with or without propofol incubation. Error bars indicate SD (n = 6). Two-way ANOVA revealed significant effects of both propofol (P = 0.0003) and the concentration of extracellular Ca2+ (P < 0.0001). There was also a significant interaction between the presence of propofol and the concentration of extracellular Ca2+ (P < 0.0001). *Significant (P < 0.05) differences based on post hoc Bonferroni test. n.s. indicates not significant at P = 0.05 level.
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