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CARDIOVASCULAR ANESTHESIA

Propofol Inhibits Volume-Sensitive Chloride Channels in Human Coronary Artery Smooth Muscle Cells

Takako Masuda, MD^*, Yoshinobu Tomiyama, MD^*, Hiroshi Kitahata, MD^*, Yasuhiro Kuroda, MD, and Shuzo Oshita, MD^*

*Department of Anesthesiology and Division of Intensive Care and Critical Care Medicine, Tokushima University School of Medicine, Tokushima, Japan

Address correspondence and reprint requests to Yoshinobu Tomiyama, MD, Department of Anesthesiology, Tokushima University School of Medicine, 3-18-15 Kuramoto, Tokushima 770-8503, Japan. Address e-mail to totomi{at}clin.med.tokushima-u.ac.jp

	Abstract

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Volume-sensitive chloride channels (VSCC) play an important role in regulation of cell volume and electrical activity. Activation of vascular smooth muscle VSCC causes smooth muscle depolarization and contraction. We investigated the effects of propofol on VSCC in cultured human coronary artery smooth muscle cells by using the chloride-sensitive dye 6-methoxy-N-ethylquinolinium (MEQ). To activate VSCC, cells were superfused for 2 min with hypotonic gluconate solutions and then potassium thiocyanate solution. The percentage reduction in MEQ fluorescence during 60 s in the presence of potassium thiocyanate was measured and used as an index of VSCC activity. 5-Nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), a well characterized chloride channel blocker, and propofol were dissolved in hypotonic gluconate solution to test their effect on VSCC activity. The reduction in fluorescence was inversely related to osmolality, indicating that activation of VSCC is osmolality dependent. Hypotonic gluconate solution (210 mOsm/kg H₂O) reduced fluorescence by 38.9% ± 2.6% of the baseline value. The reduction in fluorescence was dose-dependently inhibited by NPPB. Propofol at 0.3, 1, 3, 10, 30, and 100 µg/mL significantly inhibited the reduction in fluorescence to 23.6% ± 4.8%, 19.7% ± 7.4%, 18.2% ± 3.5%, 17.6% ± 5.0%, 15.8% ± 3.1%, and 10.3% ± 3.9% of the baseline value, respectively. Our results indicate that propofol inhibits VSCC in a dose-dependent manner in human coronary artery smooth muscle cells.

IMPLICATIONS: Propofol inhibits human coronary artery smooth muscle volume-sensitive chloride channels in a dose-dependent manner.

	Introduction

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Volume-sensitive chloride channels (VSCC) are ubiquitous in mammalian cells, where they regulate cell volume, electrical activity, intracellular pH, immunological responses, cell proliferation, and differentiation (1–3). It is possible that VSCC are active over a range of volume states that may include basal isotonic conditions, and they may play a role even in the absence of pathologic cell swelling (1). In vascular smooth muscle, VSCC play a critical role in electrophysiology (4–6). Activation of vascular smooth muscle VSCC results in Cl^- efflux and depolarization, which opens voltage-gated Ca²⁺ channels, increasing intracellular Ca²⁺ and leading to contraction (3,7). VSCC may contribute to stretch-induced constriction of vascular smooth muscle (6,8), although there is some controversy over this issue (9,10). In addition, the endothelium may regulate vascular tone in some vessels by inhibiting VSCC (11,12).

Many anesthetics reduce vascular tone. However, very little is known about anesthetic effect on VSCC activity. Blockade of VSCC activity in vascular smooth muscle will lead to vasodilation. Propofol, 2,6-diisopropyl phenol, is a short-acting, potent IV anesthetic that causes endothelium-independent vasodilatation (13,14), as well as attenuation of myogenic responses (15). The common structural basis for VSCC-blocking reagents is a phenol with hydrophobic groups, such as short alkyl chains or an additional phenyl ring (16). Because propofol is a phenol derivative, we hypothesized that propofol could block VSCC. In this study, we investigated the effects of propofol on VSCC in cultured human coronary artery smooth muscle cells by using fluorescence measurements.

	Methods

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Human coronary artery smooth muscle cells, which were certified virus free and a pure cell population on the basis of -actin staining, were obtained from Cell Application, Inc. (San Diego, CA). The cells were routinely maintained in cell culture medium (Cell Application, Inc.) at 37°C in a humidified atmosphere containing 5% CO₂. Third- to sixth -passage cultures were then seeded onto glass-bottomed culture dishes (MatTek Corp., Ashford, MA) and allowed to reach subfluence in 5–7 days.

Chloride-free Ringer’s solution was obtained by substituting chloride salts with gluconate. The composition of isotonic gluconate solution (Iso290) was (mM): Na-gluconate 135, K-gluconate 5.4, MgSO₄ 0.8, Ca-gluconate 1.2, NaH₂PO₄ 1, glucose 5.5, and Tris (hydroxymethyl) aminomethane 5, with pH adjusted to 7.3 (measured osmolality, 292 ± 7 mOsm/kg H₂O). Hypotonic gluconate solution was made by reducing Na-gluconate to 110, 91, 60, 35, and 15 mM, designated Hypo240, Hypo210, Hypo160, Hypo100, and Hypo60, respectively. The measured osmolality for each solution was 245 ± 6 mOsm/kg H₂O, 207 ± 6 mOsm/kg H₂O, 156 ± 5 mOsm/kg H₂O, 102 ± 4 mOsm/kg H₂O, and 60 ± 3 mOsm/kg H₂O (n = 6 in each group), respectively. The composition of potassium thiocyanate (KSCN) solution was KSCN 150 mM (measured osmolality, 280 ± 9 mOsm/kg H₂O). Osmolality was measured by the freezing-point technique (Osmostat OM-6040; Kyoto Diichi Kagaku, Kyoto, Japan). 5-Nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), a well characterized chloride channel blocker, and propofol were dissolved in Hypo210 from stock solutions in dimethyl sulfoxide. The final concentration of dimethyl sulfoxide was <0.1%, which does not affect Cl^- currents (8).

To facilitate loading of cells, the halide-sensitive fluorescent dye 6-methoxy-N-ethylquinolium iodide (MEQ) was reduced to cell-permeable dihydro-MEQ as described previously (17). Briefly, MEQ (5 mg/0.1 mL; 16 µM) was reduced by adding sodium borohydride (12% in H₂O; 32 µM) under a stream of nitrogen in the dark. After the reaction was completed (30 min), the dihydro-MEQ oil was extracted with ethyl acetate (0.5 mL), and extracts were dried with anhydrous MgSO₄ (100 mg). Dihydro-MEQ was stored at -80°C until use. Before each experiment, dihydro-MEQ was resuspended in Earle’s solution and used immediately.

For loading, cells were incubated in dihydro-MEQ (5 µM) solution at 37°C (5% CO₂). After 15 min of loading, cells were washed with Earle’s solution and incubated in Earle’s solution for another 15 min before measurement. Once loaded in cells, dihydro-MEQ is converted by intracellular oxidation to MEQ, which is retained within the cells.

A cell-perfusion system was designed to measure cell fluorescence in response to rapid solution changes by using an inverted epifluorescence microscope (Eclipse TS100; Nikon, Tokyo, Japan). Solutions were infused and aspirated through stainless-steel tubing into glass-bottomed culture dishes. The exchange volume of the glass bottom was approximately 150 µL. Cell-perfusion solutions were maintained at constant temperature by enclosing the perfusion solution tubing in a circulating-water jacket.

Excitation of MEQ was obtained from a xenon lamp (50 W; Nikon) filtered at 365/10 nm and reflected to the microscope objective (10x, CFI Plan Fluor ELWD 10xC; Nikon) by a dichroic mirror centered at 400 nm. Photobleaching was minimized by using a 10x microscope objective and neutral density filters: 2.5–3.5 optical density (OD). Cell fluorescence was collected by the objective, passed to a 400-nm-long path filter, and directed to a digital cooled black and white charge-coupled device camera (ORCA; Hamamatsu Photonics, Hamamatsu, Japan). Fluorescence images were acquired at 15-s intervals by using an image processing system (Aquacosmos; Hamamatsu Photonics) and stored in a computer. To further limit photobleaching, excitation was manually synchronized with sampling. Fluorescence images were analyzed for average pixel intensities of regions of interest (ROI). These ROI were selected manually with a maximum of approximately five cells per field. The responses of all selected cells were averaged to yield a response per dish.

VSCC possess several common biophysical and pharmacological properties, including activation by hypotonic solutions, an anion selectivity sequence of thiocyanate (SCN^-) > I^- = NO₃^- > Br^- > Cl^- > F^- > gluconate, and sensitivity to blockade by NPPB (2). We used a modified method of fluorescence measurements of VSCC activity (17). The cells were continuously superfused with a gluconate solution at 35°C on glass-bottomed culture dishes at 4 mL/min for 2 min. MEQ fluorescence is quenched by anions, and fluorescence intensity is inversely related to intracellular anions (17). Replacement of chloride salts with gluconate is necessary for maintaining a maximum initial fluorescence of MEQ (17). Gluconate is less permeable than Cl^- in VSCC (17). At the time points indicated (arrows in Fig. 1), the gluconate solution was changed to a KSCN solution. The extracellular KSCN solution was used to confirm anion passage through VSCC, because SCN^- is more permeable to VSCC than Cl^- and can quench MEQ fluorescence more effectively than can Cl^-. A large concentration of SCN^- was used to enhance the signal/noise ratio (17). Although the osmolality of the KSCN solution was almost isotonic, this was not expected to inactivate the VSCC completely during measurements because, once activated by hypotonic solution, Cl^- current persists in an activated state for at least 60 s even under isotonic conditions (8,17). The percentage change in MEQ fluorescence is calculated as follows: equation

View larger version (19K): [in this window] [in a new window]   Figure 1. Time course of fluorescence measurements and calculation of change in fluorescence. Iso290 = representative 6-methoxy-N-ethylquinolinium (MEQ) fluorescence response in the group that was treated with a 290 mOsm/kg H2O isotonic gluconate solution; Hypo210 = representative MEQ fluorescence response in the group that was treated with a 210 mOsm/kg H2O hypotonic gluconate solution; F0 = fluorescence before beginning KSCN perfusion; FSCN = fluorescence 60 s after beginning KSCN perfusion. Cells were superfused at 35°C with solutions continuously at 4 mL/min for 2 min. At the points indicated (arrow), the gluconate solution was changed to a potassium thiocyanate (KSCN) (150 mM) solution.

To investigate the effects of osmolality on anion movement across the cell membrane, 36 dishes were randomly assigned to 6 groups of different osmolality: 290 mOsm/kg H₂O (Iso290), 250 mOsm/kg H₂O (Hypo250), 210 mOsm/kg H₂O (Hypo210), 150 mOsm/kg H₂O (Hypo150), 100 mOsm/kg H₂O (Hypo100), and 60 mOsm/kg H₂O (Hypo60) (n = 6 in each group). To investigate the effects of NPPB on VSCC, 16 dishes treated with Hypo210 were randomly assigned to 4 groups of different NPPB concentrations: 0, 50, 100, and 200 µM (n = 4 in each group). To investigate the effects of propofol on VSCC, 48 dishes treated with Hypo210 were randomly assigned to 8 groups of different propofol concentrations: 0, 0.1, 0.3, 1, 3, 10, 30, and 100 µg/mL (n = 6 in each group).

MEQ was purchased from Molecular Probes (Eugene, OR). NaH₂PO₄ was purchased from Merck (Darmstadt, Germany). All other drugs were purchased from Sigma (St. Louis, MO).

All results are expressed as mean ± SD. Values were compared by using analysis of variance with the Scheffé post hoc test. A value of P < 0.05 was accepted as significant.

Results
MEQ was used to measure the change in fluorescence proportional to the anion permeability in single human coronary artery smooth muscle cells. Representative MEQ fluorescence responses are shown in Figure 1. Replacement of chloride salts with gluconate in perfusion solution significantly increased the MEQ signal during the perfusion with gluconate solutions from 427 ± 66 (arbitrary unit) to 472 ± 70 (11% ± 5%) and from 465 ± 86 to 558 ± 153 (20% ± 15%) in Iso290 and Hypo210, respectively (P < 0.05 versus the value at the beginning of perfusion), suggesting an anion efflux in isosmotic and hypotonic conditions. Quenching of the MEQ signal was determined by adding SCN^- to the extracellular fluid after exposing the cells to isosmotic and hypotonic media. Under isosmotic conditions in the presence of KSCN, only minute quenching of the MEQ signal was obtained. Quenching of the MEQ signal increased in parallel with decreasing osmolalities, and all changes were significantly different from those with Iso290 (Fig. 2). In cells with VSCC activated by the Hypo210 solution, the reduction in fluorescence was significantly attenuated in a concentration-dependent fashion by NPPB (Fig. 3).

View larger version (14K): [in this window] [in a new window]   Figure 2. Effects of osmolality on change of methoxy-N-ethylquinolinium fluorescence in human coronary smooth muscle cells (n = 6). Values are expressed as mean ± SD. *P < 0.05 versus the value of a 290 mOsm/kg H2O isotonic gluconate solution.

View larger version (13K): [in this window] [in a new window]   Figure 3. Effects of 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) on hypotonicity-induced change in methoxy-N-ethylquinolinium fluorescence. The group treated with 0 µM concentrations of NPPB was equivalent to the group treated with a 210 mOsm/kg H2O hypotonic gluconate solution (Hypo210) alone. NPPB significantly attenuated the reduction in fluorescence by Hypo210 in a concentration-dependent fashion (n = 4). Values are expressed as mean ± SD. * P < 0.05 versus the value of 0 µM concentrations of NPPB; #P < 0.05 versus the value of 50 µM concentrations of NPPB.

View larger version (15K): [in this window] [in a new window]   Figure 4. Effects of propofol on hypotonicity-induced change in methoxy-N-ethylquinolinium fluorescence. The group treated with 0 µg/mL concentrations of propofol was equivalent to the group treated with a 210 mOsm/kg H2O hypotonic gluconate solution (Hypo210) alone. Propofol significantly attenuated the reduction in fluorescence by Hypo210 (n = 6). Values are expressed as mean ± SD. *P < 0.05 versus the value of 0 µg/mL concentrations of propofol; #P < 0.05 versus the value of 0.1 µg/mL concentrations of propofol. P < 0.05 versus the value of 0.3 and 1 µg/mL concentrations of propofol.

	Discussion

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In this study, we showed, using MEQ-based methods, that propofol inhibition of VSCC is dose dependent. During clinical anesthesia, total and free plasma propofol concentrations differ significantly. Park et al. (19) estimated that total propofol blood concentrations range from 1 to 20 µg/mL during clinical anesthesia. The free plasma concentration of propofol is 0.6 µg/mL (3.4 µM) or less (19) because 97%–99% of propofol is bound to plasma proteins. We previously reported that actual propofol concentrations in the superfusate of our experimental system were 0.4 ± 0.1 µg/mL, 1.3 ± 0.2 µg/mL, 5.6 ± 0.4 µg/mL, 16.7 ± 0.6 µg/mL, and 60.1 ± 3.6 µg/mL at total concentrations of 1, 3, 10, 30, and 100 µg/mL, respectively (20). Thus, in our study, propofol significantly inhibited VSCC at a clinically relevant concentration.

We found that hypotonic stimulation reduced MEQ fluorescence in an osmolality-dependent manner. Previous studies based on the patch-clamp technique have shown that hypotonic stimulation activates volume-sensitive chloride currents in smooth muscle cells (5,8). Although our estimates of VSCC activity based on MEQ fluorescence are qualitative (17,21,22), the half-maximal activation of VSCC occurred in our study at approximately 171 mOsm/kg H₂O. This result is similar to the half-maximal osmolal activation of endothelial cell VSCC observed with the patch-clamp method (23). In our study, 50–200 µM NPPB significantly inhibited the reduction in fluorescence in a concentration-dependent fashion, and this was similar to the concentration-dependent effect of NPPB on VSCC with the patch-clamp method (2). We think that this is clear evidence that the reduction in fluorescence was mediated by activation of VSCC. Although available pharmacological blockers are not entirely selective for VSCC (2), the effects of cation movement could be ignored because our method measured only changes in intracellular anion concentration. Thus, on the basis of this information, we conclude that hypotonicity activated VSCC-dependent anion movement, which was inhibited by NPPB.

Activation of VSCC (4,6) and a swelling-activated nonselective cation channel (9,10) may be involved in the mechanism by which pressure causes depolarization. Activation of VSCC produces depolarization because the Cl^- equilibrium potential (approximately -20 to -30 mV) is substantially more positive than the resting membrane potential (approximately -50 to -75 mV) (7). Another important Cl^- channel in vascular smooth muscle is the Ca²⁺-activated Cl^- channel (7). Activation of the swelling-activated nonselective cation channel may result in Ca²⁺ influx and opening of Ca²⁺-activated Cl^- channels. However, the Cl^- channels involved in the myogenic response were insensitive to niflumic acid, an effective blocker of Ca²⁺-activated Cl^- channels (4,24). In addition, blocking Ca²⁺ current and/or reducing Ca²⁺ appears not to explain the inhibition of Cl^- efflux, because nimodipine, a selective Ca²⁺ channel antagonist, inhibited myogenic tone but had no effect on Cl^- flux (6). Nelson et al. (4) reported that pressure-induced depolarization of cerebral arteries occurs in the presence of Ca²⁺ channel blockers such as nisoldipine and diltiazem, which reduce intracellular Ca²⁺ concentrations to less than those required for Ca²⁺-activated chloride channel activity. Furthermore, Nagase et al. (25) reported that propofol does not attenuate the induction of Ca²⁺-activated Cl^- currents by the intracellular injection of CaCl₂, indicating that propofol does not affect Ca²⁺-activated Cl^- channels in Xenopus oocytes. Although species- and expression system-dependent differences from human coronary artery smooth muscle cells might be present, those facts suggest that in our study, propofol reduced anion entry by blockade of VSCC and not by Ca²⁺-activated Cl^- channels.

Chloride-sensitive fluorescent indicators provide an approach for studying Cl^- transport that is complementary to the intracellular microelectrode and patch-clamp method (21). The potential advantages of Cl^--sensitive fluorescent indicators over other available methods are technical simplicity, noninvasiveness, high time-resolution for measurement of fast transport processes, and detection of the electroneutral movements of Cl^- (17,21).

Some limitations of this study should be pointed out. Although ion flux, as measured, is influx, this is only a result of the technique, and under normal physiologic conditions, the flux would be in the opposite direction. We have used hypotonic stress as an in vitro example of mechanical stress in this study. Welsh et al. (9) reported that a hypotonic shock elicited a functional response that was similar to myogenic control. It should be noted that the effects of propofol on VSCC were evaluated when VSCC were activated with a 210 mOsm/kg H₂O hyposmolar solution. Lang et al. (3) reported that exposure of human vascular smooth muscle cells to 25% hypotonic extracellular fluids led to a rapid depolarization of 13 mV. An increase of transmural pressure from 0 to 100 mm Hg induced a 16-mV membrane depolarization in cat middle cerebral artery (26). Thus, Hypo210 was selected in this study to induce a mechanical stress that was relevant to mechanical stress that occurs in vivo. Doughty and Langton (6) reported that myogenic contraction was accompanied by a temperature-dependent increase in Cl^- efflux from arteries. We performed experiments by keeping the temperature at 35°C. Differences in VSCC function may occur in vivo, where mechanisms of activation of the channel may differ. However, our data support the ability of propofol to block VSCC function.

In conclusion, we have demonstrated that propofol inhibits volume-sensitive chloride channels in a dose-dependent manner in human coronary artery smooth muscle cells. This result may provide insight into the vasodilatory effects of propofol.

	Acknowledgments

 
Supported by a Grant-in-Aid (12671476) for Scientific Research from the Japan Society for the Promotion of Science, Tokyo, Japan.

The authors thank Dr. Johnny E. Brian, Jr., Department of Anesthesia, University of Iowa, for critical reading of the manuscript.

	Footnotes

 
Presented in part at the annual meeting of the American Society of Anesthesiologists, New Orleans, LA, October 15, 2001.

	References

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a colleague Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via Web of Science (3) Citing Articles via Google Scholar Google Scholar Articles by Masuda, T. Articles by Oshita, S. Search for Related Content PubMed PubMed Citation Articles by Masuda, T. Articles by Oshita, S. Related Collections Heart Pharmacology