Involvement of Adenosine Triphosphate-Sensitive Potassium Channels in the Response of Membrane Potential to Hyperosmolality in Cultured Human Aorta Endothelial Cells

Abstract

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 (K(ATP)) 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 H(2)O) containing 200 nM bis-(1,3-diethylthiobarbiturate)trimethine oxonol and exposed to 315 and 345 mOsm/kg H(2)O hyperosmotic medium sequentially in the presence and absence of 1 muM glibenclamide, a well-known K(ATP) channel blocker. Hyperosmotic mannitol significantly induced hyperpolarization of the endothelial cells, which was prevented by 1 microM glibenclamide (n = 6). Estimated changes of membrane potential at 315 and 345 mOsm/kg H(2)O 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 muM glibenclamide. In conclusion, K(ATP) channels may participate in hyperosmotic mannitol- and sucrose-induced hyperpolarization, but not in hypertonic saline-induced hyperpolarization in cultured human aorta endothelial cells.

Calibration procedures for the quantitative determination of membrane potential in human cells using anionic dyes

“…It also seems not to alter the membrane potential in whole cells in Na‐free K‐NMDG media (this study, data not shown). Still, the commonly used approach of Method 3A has been to add gramicidin . In addition, it has been assumed that [K + ] i stays constant irrespective of changes in [K + ] o and that E K reflects membrane potential .…”

“…With K‐Na media (Method 1) and Na‐NMDG media (Method 2), calculation was performed according to the GHK equation: where p is the ratio of the membrane permeabilities for Na + and K + ( P Na / P K ) assumed to be 0.05 in untreated cells (Method 1) or 1 in gramicidin‐treated cells (Method 2). Comparable with other studies , the intracellular concentrations [K + ] i and [Na + ] i were taken as 140 and 10 m M , respectively. In calibrations using Na‐free K‐NMDG media (Method 3), the apparent membrane potential was calculated according to the Nernst potential for potassium ( E K ) as a special case of the GHK equation: where [K + ] i was assumed to be 140 m M as well.…”

“…(Method 1) or 1 in gramicidin-treated cells (6,7,9,10,(13)(14)(15)17,19,21,23,24) (Method 2). Comparable with other studies (6,7,9,10,13,14,16,19,20,22,24,60,61), the intracellular concentrations [K 1 ] i and [Na 1 ] i were taken as 140 and 10 mM, respectively. In calibrations using Na-free K-NMDG media (Method 3), the apparent membrane potential was calculated according to the Nernst potential for potassium (E K ) as a special case of the GHK equation:…”

“…Still, the commonly used approach of Method 3A has been to add gramicidin (8,12,16,20,22,26). In addition, it has been assumed that [K 1 ] i stays constant irrespective of changes in [K 1 ] o and that E K reflects membrane potential (8,12,16,20,22,26). Steinberg et al (21) modified this method by media containing a residual [Na 1 ] o of 5 mM and calculated the membrane potential according to the GHK equation.…”

“…As this method is quite laborious, the number of measuring values within a reasonable time is quite low. In a lot of studies using spectrofluorimetry , microscopic imaging , or flow cytometry , calibration was usually based on applying a voltage ramp by varying concentrations of extracellular sodium or potassium with isosmolar substitution of the respective alkali ion for choline chloride or N ‐methyl‐ D ‐glucamine (NMDG) in the presence of gramicidin (anionic dyes) or valinomycin (cationic dyes). In spectrofluorimetric and fluorescence imaging experiments, changes in external potassium concentration were also performed by sequential additions of KCl .…”