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Anesth Analg 2005;100:419-426 © 2005 International Anesthesia Research Society doi: 10.1213/01.ANE.0000143350.82645.5B
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Abstract |
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The membrane potential of endothelial cells is an important determinant of endothelial functions, including regulation of vascular tone. We investigated whether adenosine triphosphate-sensitive potassium (KATP) channels were involved in the response of membrane potential to hyperosmolality in cultured human aorta endothelial cells. The voltage-sensitive fluorescent dye, bis-(1,3-diethylthiobarbiturate)trimethine oxonol, was used to assess relative changes in membrane potential semiquantitatively. To investigate the effect of mannitol-, sucrose-, and NaCl-induced hyperosmolality on membrane potential, cells were continuously perfused with Earle’s balanced salt solution (285 mOsm/kg H2O) containing 200 nM bis-(1,3-diethylthiobarbiturate)trimethine oxonol and exposed to 315 and 345 mOsm/kg H2O hyperosmotic medium sequentially in the presence and absence of 1 µM glibenclamide, a well-known KATP channel blocker. Hyperosmotic mannitol significantly induced hyperpolarization of the endothelial cells, which was prevented by 1 µM glibenclamide (n = 6). Estimated changes of membrane potential at 315 and 345 mOsm/kg H2O were 13 ± 8 and 21 ± 8 mV, respectively. Hypertonic sucrose induced similar changes. However, although hypertonic saline also significantly induced hyperpolarization of the endothelial cells (n = 6), the hyperpolarization was not prevented by 1 µM glibenclamide. In conclusion, KATP channels may participate in hyperosmotic mannitol- and sucrose-induced hyperpolarization, but not in hypertonic saline-induced hyperpolarization in cultured human aorta endothelial cells.
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Introduction |
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Hyperosmotic mannitol (1,2) and hypertonic saline (3) are often used clinically for the management of increased intracranial pressure and hypovolemic shock, respectively. In addition, tissue osmolality increases during ischemia and exercise (4,5). Infusion of osmotic agents can cause vasodilation (3,6). Increased tissue osmolality also has an important role in regional vasodilation (4,5). There is evidence to suggest that the response could be mediated by membrane hyperpolarization of vascular endothelial cells (4). Endothelial hyperpolarization may cause vasodilation by transferring the membrane hyperpolarization to the underlying smooth muscle cells via gap junctions (4,7–9) or by stimulating the Ca2+-dependent release of vasoactive compounds, such as nitric oxide and prostacyclin, from endothelial cells (3,8–10). Mechanisms of hyperosmolality-induced endothelial hyperpolarization have not yet been elucidated. Some evidence suggests that hyperosmolar glucose or sucrose solution may activate adenosine triphosphate-sensitive potassium (KATP) channels leading to hyperpolarization of vascular endothelial cells (4,5). However, direct evidence for the participation of KATP channels in hyperosmolality-induced endothelial hyperpolarization has not yet been obtained. Indeed, another report suggests that KATP channel activating agents may cause hyperpolarization of endothelial cells, which may occur through gap junction-mediated transfer of smooth muscle hyperpolarization to the endothelium (11). We investigated a relationship between activation of the KATP channels and hyperpolarization in the absence of vascular smooth muscle. Furthermore, although both hypertonic saline and hyperosmotic sucrose increase myocardial contractility, the mechanisms are different (12). The sodium ion itself has a key role in this improvement of myocardial contractility through a specific mechanism involving Na+-Ca2+ exchange (12). Na+-Ca2+ exchange is not involved in the sucrose-induced improvement of contractility (12). To better understand the mechanism by which hyperosmolality affects membrane potential, we investigated the effects of mannitol-, sucrose-, and NaCl-induced hyperosmolality on membrane potential in the presence or absence of glibenclamide, a well known KATP channel blocker, in cultured human aorta endothelial cells.
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Methods |
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Cell Culture
Human aorta endothelial cells, which were certified to be virus-free and pure cell populations on the basis of staining patterns for
-actin, were obtained from Cell Application, Inc. (San Diego, CA). The cells were routinely maintained in cell culture medium (Cell Application) at 37°C in a humidified atmosphere containing 5% CO2. The third to sixth passage cultures were then seeded onto glass-bottom culture dishes (MatTek Corp., Ashford, MA) and allowed to reach subconfluence in 5–7 days.
Reagents
Earle’s balanced salt solution (measured osmolality, 285 ± 1 mOsm/kg H2O) was used and its composition was as follows (in mM): NaCl 116.4, KCl 5.4, CaCl2 1.8, MgSO4 0.8, NaHCO3 26.2, NaH2PO4 1, and d-glucose 5.6 with pH adjusted to 7.3. Hyperosmotic solutions were obtained by adding mannitol, sucrose, or NaCl to the Earle’s balanced salt solution. Measured osmolality values of the hyperosmolality solutions were 315 ± 2 mOsm/kg H2O in 315 mOsm/kg H2O conditions and 344 ± 3 mOsm/kg H2O in 345 mOsm/kg H2O conditions, respectively. Osmolality was measured by the freezing point technique (Osmostat OM-6040; Kyoto Diichi Kagaku, Kyoto, Japan). The pH of the solutions was kept at 7.35 throughout the study by bubbling the solutions with 5% CO2 and 95% O2. Glibenclamide, a KATP channel blocker, levcromakalim, a KATP channel opener, and a voltage-sensitive fluorescent dye, bis-(1,3-diethylthiobarbiturate)trimethine oxonol (bis-oxonol) were dissolved in Earle’s balanced salt solution from stock solutions in dimethyl sulfoxide (the final solvent concentration was 0.02%, 0.001%, and 0.001%, respectively).
Cell Perfusion System
A cell perfusion system was designed to measure cell fluorescence in response to rapid solution changes. The system used an inverted epifluorescence microscope (Eclipse TS100; Nikon, Tokyo, Japan). Solutions were infused and aspirated through stainless steel tubing onto the glass bottom of the culture dishes. The exchange volume of the glass bottom was approximately 150 µL. Cell perfusion solutions were maintained at a constant temperature by enclosing the perfusion tubing in a circulating water jacket.
Measurement of Membrane Potential
We used bis-oxonol to assess relative changes in the membrane potential of single (subconfluent) cells semiqualitatively as described in our previous report (13). This dye has been used previously to determine changes in membrane potential, including endothelial cells (8,14,15). When the cell membrane is depolarized, the dye partitions into the membrane leading to an increase in the measured fluorescence. In brief, the cells were continuously superfused with Earle’s balanced salt solution containing 200 nM bis-oxonol at 36°–37°C. Excitation of bis-oxonol was obtained from a Xenon lamp (50 W; Nikon) filtered at 450–490 nm and reflected on the microscope objective (x10, CFI Plan Fluor ELWD 10 x C; Nikon). Cell fluorescence was collected by the objective, passed to a 520-nm-long path filter, and directed to a cooled digital black-and-white charge coupled device camera (ORCA; Hamamatsu Photonics, Hamamatsu, Japan). Fluorescence images were acquired at 60-s intervals by means of a computer-controlled shutter (Filter Exchanger; Hamamatsu Photonics) and an image processing system (Aquacosmos; Hamamatsu Photonics). Fluorescence images were analyzed for average pixel intensities of regions of interest. Each region of interest contained 1–6 cells. These regions were selected manually with a maximum of 6 regions per field. The responses of all selected cells were averaged to yield one response per dish. Background values (windows of identical area placed beside the cells) were always subtracted. Once bis-oxonol fluorescence attained equilibrium (approximately 30 min), the effects of the pharmacological agents and osmolality on membrane potential were determined. Bis-oxonol fluorescence was normalized relative to the value approximately 30 min after initiation of superfusion (baseline; in percent).
Experimental Protocols
To investigate the effects of 1 µM glibenclamide on the response of membrane potential to mannitol-induced hyperosmolality, 18 dishes were randomly assigned to 3 experimental protocols: 1) cells were superfused with isosmotic solution throughout the experiments (isosmotic time control, n = 6), 2) cells were sequentially exposed to 315 and 345 mOsm/kg H2O solution in the absence of glibenclamide (hyperosmolality, n = 6), and 3) cells were sequentially exposed to 315 and 345 mOsm/kg H2O solution in the presence of glibenclamide (hyperosmolality + glibenclamide, n = 6). Dimethyl sulfoxide concentration in solutions in the isosmotic time control group and hyperosmolality group were increased to 0.02% at the corresponding time of glibenclamide application in the hyperosmolality + glibenclamide group. The values at 30 and 45 min after the baseline point were used for statistical analysis. In the second series of experiments, the experiments were repeated with the identical protocol except that NaCl was used as the hyperosmotic stimulus. The values of isosmotic time control in the first series of experiments were used for the second. To exclude the specific effects of mannitol, after the previous two series of experiments were finished, we investigated the effects of sucrose, another non-ionic hyperosmotic agent.
As a test for the presence of KATP channels in cultured human aortic endothelial cells, we investigated the effects of levcromakalim on membrane potential. The cells were exposed to levcromakalim in the presence or absence of glibenclamide. The values at 20 and 40 min after the baseline point were used for statistical analysis.
Calibration of Bis-Oxonol Fluorescence
After all experiments were finished, calibration of bis-oxonol fluorescence was performed using the Na+ ionophore gramicidin in Na+-free physiological salt solution (13). In brief, in the presence of gramicidin (2 µg/mL), the transmembrane Na+ concentration gradient is zero, and membrane potential is approximately equal to K+ equilibrium potential, which at 37°C is determined by the Nernst equation. The intracellular potassium concentration was assumed to be 137 mM (16). The addition of gramicidin with various concentrations of K+ to the endothelial cells altered the cell membrane potential, thereby altering fluorescence (Fig. 1A). Extracellular potassium concentrations used in this study were 9, 17, and 25 mM, such that membrane potentials varied among –73, –56, and –45 mV as calculated by the Nernst equation. We studied all possible sequences of extracellular potassium concentrations (n = 6) (Fig. 1A). The reported resting membrane potentials in endothelial cells without tone were approximately –50 mV (8,11,14). The calculated resting membrane potentials in cultured human aorta endothelial cells in our calibration study in Na-free physiological salt solution were –48 ± 17 mV. Therefore, bis-oxonol fluorescence was normalized relative to the value at the end of 17 mM (percent). A calibration curve of the relation of cell-associated bis-oxonol fluorescence with membrane potential was constructed and used to estimate the changes in membrane potential (Fig. 1B). A decrease in fluorescence by 1% corresponds to a hyperpolarization of approximately 0.64 mV as calculated from the mean calibration curve.
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Materials
Glibenclamide, levcromakalim, bis-oxonol, and NaH2PO4 were purchased from Wako (Osaka, Japan), TOCRIS (Ellisville, MO), Molecular Probes (Eugene, OR) and Merck (Darmstadt, Germany), respectively. All other drugs were purchased from Sigma (St. Louis, MO).
All results were expressed as means ± sd. Values were compared using analysis of variance with the Scheffé post hoc test. A value of P < 0.05 was accepted as significant.
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Results |
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With infusion of the bis-oxonol and continuous monitoring, fluorescence from the endothelial cells increased and attained a steady level after 20–30 min.
The effects of mannitol-induced hyperosmolality on membrane potential in the presence and absence of glibenclamide were studied. Results obtained from representative cells are illustrated in (Figure 1A. The relative bis-oxonol fluorescences at 315 and 345 mOsm/kg H2O in the hyperosmolality (mannitol) group were significantly different from those in the isotonic time control group (Fig. 2B), indicating membrane hyperpolarization via mannitol-induced hyperosmolality. Hyperpolarizations of the membrane potential were 13 ± 8 and 21 ± 8 mV, respectively, as calculated from the calibration curve obtained with cultured human aortic endothelial cells. Glibenclamide did not alter the resting membrane potential. The relative bis-oxonol fluorescences in the hyperosmolality (mannitol) + glibenclamide group were significantly different from those in the hyperosmolality (mannitol) group (Fig. 2B), indicating participation of the KATP channel in hyperosmotic mannitol-induced hyperpolarization. The estimated hyper-polarizations of the membrane potential in the hyperosmolality (mannitol) + glibenclamide group were 2 ± 4 and 1 ± 3 mV at 315 and 345 mOsm/kg H2O mannitol, respectively. The relative bis-oxonol fluorescences in the hyperosmolality (mannitol) + glibenclamide group were not significantly different from those in the isotonic time control group.
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The effects of NaCl-induced hyperosmolality on membrane potential in the presence and absence of glibenclamide were studied. Results obtained from representative cells are illustrated in (Figure 3A. The relative bis-oxonol fluorescences at 315 and 345 mOsm/kg H2O in the hyperosmolality (NaCl) group were significantly different from those in the isotonic time control group (Fig. 3B), indicating membrane hyperpolarization via NaCl-induced hyperosmolality. The estimated hyperpolarizations of the membrane potential were 15 ± 6 and 19 ± 8 mV, respectively. Relative bis-oxonol fluorescences at 315 and 345 mOsm/kg H2O in the hyperosmolality (NaCl) + glibenclamide group were not significantly different from those in the hyperosmolality (NaCl) group (Fig. 3B), indicating absence of the KATP channel’s participation in hyperosmotic saline-induced hyperpolarization. The estimated hyperpolarizations of the membrane potential in the hyperosmolality (NaCl) + glibenclamide group were 13 ± 7 and 23 ± 9 mV, respectively
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The effects of sucrose-induced hyperosmolality on membrane potential in the presence and absence of glibenclamide were studied. The results obtained from representative cells are illustrated in (Figure 3A. The relative bis-oxonol fluorescences at 315 and 345 mOsm/kg H2O in the hyperosmolality (sucrose) group were significantly different from those in the isotonic time control group (Fig. 4B), indicating membrane hyperpolarization via sucrose-induced hyperosmolality. The estimated hyperpolarizations of the membrane potential were 9 ± 2 and 17 ± 9 mV, respectively. The relative bis-oxonol fluorescences in the hyperosmolality (sucrose) + glibenclamide group were significantly different from those in the hyperosmolality (sucrose) group (Fig. 4B), indicating participation of the KATP channel in hyperosmotic sucrose-induced hyperpolarization. The estimated hyperpolarizations of the membrane potential in the hyperosmolality (sucrose) + glibenclamide group were 0 ± 8 and 1 ± 13 mV, respectively. The relative bis-oxonol fluorescences in the hyperosmolality (sucrose) + glibenclamide group were not significantly different from those in the isotonic time control group.
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The application of 1 µM levcromakalim to endothelial cells caused a sustained decrease in fluorescence (Fig. 5). The estimated membrane-potential change was 20 ± 10 mV. The subsequent application of 1 µM levcromakalim plus 1 µM glibenclamide resulted in the slow recovery of the resting potential, indicating the presence of KATP channels in the cultured human aorta endothelial cells.
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Discussion |
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The main findings of the present study were that all investigated hyperosmolar solutions hyperpolarized the cultured human aorta endothelial cells. Although the activation of KATP channels may participate in hyperosmolar mannitol- and hyperosmolar sucrose-induced membrane hyperpolarization, it may not participate in hypertonic saline-induced membrane hyperpolarization.
We demonstrated that hyperosmolar solutions caused membrane hyperpolarization in human aorta endothelium cells in this study. The magnitudes of hyperpolarization were similar among the three groups. Endothelial hyperpolarization results in vasodilation as described in the Introduction. Thus, these findings coincide well with a previous report that hyperosmotic mannitol and hypertonic saline dilated cerebral arteries to a similar degree, and the effect seemed to be attributable solely to an increase in osmolality (6). Very few studies have evaluated the hyperosmolality-induced hyperpolarization in endothelial cells directly (9,17). Voets et al. (9) reported that cultured bovine pulmonary artery endothelial cells with membrane potentials more negative than the reversal potential for chloride were hyperpolarized by 15 mV with the addition of 100 mM mannitol. In contrast, in isolated rat aorta endothelial cells, the addition of 100 mM sucrose evoked a small (<5 mV) depolarization of the endothelial membrane potential (17). Estimated changes in membrane potential by the addition of 30 and 60 mM mannitol were approximately 13 and 21 mV in this study. The relatively large change in our experiment might be explained by a difference of vessel, species, or experimental conditions.
Hyperosmotic mannitol-induced membrane hyperpolarization was inhibited by 1 µM glibenclamide in this study. Most evidence suggests that glibenclamide selectively blocks KATP channels at a concentration of 1 µM (18). In addition, Ishizaka and Kuo (4) reported that Ca2+-activated or inward rectifier potassium channels have no effect on hyperosmolality-induced vasodilation. And activation of Ca2+-activated potassium channels induced only a small hyperpolarization (approximately 2.5 mV) in endothelial cells (11). Thus, our results suggest that KATP channels participate in hyperosmotic mannitol-induced membrane hyperpolarization. These findings coincide well with a previous report that non-ionic hyperosmolar solutions may activate KATP channels in endothelial cells using diameter measurements in isolated cannulated and pressurized porcine coronary arterioles (4) as well as skeletal muscle arterioles (5). KATP channels have been found on vascular endothelial cells (10) and activation of these channels hyperpolarizes the endothelial cells (8,14). An experimental concern is that endothelial cells may undergo significant changes in culture (8). We demonstrated that 1 µM levcromakalim, a KATP channel opener, hyperpolarized the human aorta endothelial cells by 20 mV and the effects of levcromakalim could be reversed by an application of 1 µM glibenclamide in this study. Most evidence suggests that 1 µM levcromakalim selectively activates KATP channels at this concentration (18). These findings suggest that our cultured cells expressed KATP channels. It is of note that the potency of 1 µM levcromakalim to cause hyperpolarization of the endothelial cells was close to the data in cultured human umbilical veins (14 mV) (14) and in freshly isolated coronary capillaries (30 mV) (8,10) induced by various K+ channel openers. The mechanisms for the non-ionic hyperosmolar solution-induced activation of KATP channels have not been elucidated.
Many channels including KATP channels can determine the endothelial membrane potential (9,15,19,20) and the effects of these channels on endothelial membrane potential are complex and multifactorial (9,19,20). For example, Doughty et al. (19) reported that hyperpolarization by an inwardly rectifying potassium current depends on the magnitude of a volume-sensitive chloride current, which can be inhibited by hyperosmolality. One possible explanation of KATP channel activation is the activation of Na+-K+-adenosine triphosphatase (ATPase) induced by regulatory volume increase after cell shrinkage (20). Regulatory volume increases are coupled with increased Na+-K+-ATPase activity (20,21). Na+-K+-ATPase activation may lead to the activation of KATP channels (22) via submembrane ATP depletion (23). However, further experiments will be required to check these possibilities and shrinkage itself may increase intracellular adenosine diphosphate concentration and/or activate other regulatory mechanisms, such as integrin-mediated mechanotransduction pathways (4). Thus, based on this information, we assumed that hyperosmotic mannitol activated KATP channels. Hyperosmotic sucrose indicates almost the same result with mannitol. Thus, these effects are not mannitol-specific effects, but common effects in non-ionic hyperosmotic agents.
Hypertonic saline also induced hyperpolarization in this study. However, KATP channels were not involved in hypertonic saline-induced membrane hyperpolarization in contrast to hyperosmotic mannitol. A major difference between hyperosmotic mannitol and hypertonic saline is their effect on intra- and/or extracellular electrolyte concentrations (12,20). If extracellular osmolality is made hypertonic by increased extracellular NaCl concentration, the increase of intracellular Cl– activity could impede a regulatory volume increase (20,21). This inhibition may result in the failure of KATP channel activation, as described in the early part of this discussion. However, KATP channel-independent hyperosmolality-induced membrane hyperpolarization is possible. Hypertonic saline causes hyperchloridemia. Cl– removal from the extracellular space or low Cl– leads to depolarization (15). Thus, although mechanisms remain to be clarified, these effects of electrolytes may participate in the difference in mechanisms.
Mannitol is used to reduce intracranial pressure and decrease brain bulk in neurosurgical patients (1,2). The administration of 2 mg/kg of 20% mannitol infused at a rate of 15 mL/min and 1 mg/kg infused over 4 minutes increases the plasma osmolality by 30 (1) and 36 mOsm/kg H2O (2), respectively. The interstitial osmolality of the myocardium increases by 20 and 40 mOsm/kg H2O within 10 and 50 minutes, respectively, after coronary occlusion in porcine hearts (24). Therefore, the osmolalities of 315 and 345 mOsm/kg H2O chosen in this study are clinically relevant.
The bis-oxonol dye system semiquantitatively captures the behavior of cell membrane-potential responsiveness to various stimuli. The bis-oxonol dye response time is relatively slow (a few seconds), which prevents measurements of precise physiological time constants associated with membrane-potential change (15). However, conventional direct measurement of membrane potential with a patch electrode or glass microelectrode has the risk of electrical and mechanical perturbation. The minimal invasiveness of the dye to the cells makes this technique attractive (15). Furthermore, the recording of the membrane potential with bis-oxonol could be especially useful for indirectly testing KATP channel activities, because the washout of cytosolic constitutes occurring during conventional whole cell measurements is avoided (8).
Limitations of this study should be clarified. Physiological blood vessels are exposed to flow (15) and intraluminal pressure (7). Those factors might alter the membrane potential of endothelial cells (15). Resting membrane potentials are important for the response in membrane potential to various stimuli in endothelial cells (19). Therefore, the response to hyperosmolality in physiological conditions might differ. We used aortic endothelial cells. We should explore findings in endothelial cells in other organs with caution. However, the maintenance of normal aorta endothelial function itself is important. The aorta is a dynamic organ, capable of almost instantaneous changes in size and elasticity (25). The elastic properties of the aorta are an important determinant of left ventricular function and coronary blood flow (25). The decrease of fluorescence with application of hyperosmolar mannitol did not reach a plateau at 315 mOsm/kg H2O. Therefore, we might underestimate the hyperpolarization at 315 mOsm/kg H2O. However, this possible underestimation does not have a major effect on the findings. Despite some limitations, our results may explain in part the mechanism by which hyperosmolality affects membrane potential and vascular tone.
In conclusion, all hyperosmotic agents induced membrane depolarization in cultured human aorta endothelial cells. Our results suggest that KATP channels participate in hyperosmotic mannitol- and sucrose-induced membrane hyperpolarization in cultured human aorta endothelial cells. Different mechanisms may participate in hypertonic saline-induced hyperpolarization.
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Footnotes |
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This study was supported by a Grant-in Aid (12671476) for Scientific Research (C) from the Japan Society for the Promotion of Science, Tokyo, Japan.
Accepted for publication August 10, 2004.
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References |
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