2,2,2-trichloroethanol activates a nonclassical potassium channel in cerebrovascular smooth muscle and dilates the middle cerebral artery

Abstract

Trichloroacetaldehyde monohydrate [chloral hydrate (CH)] is a sedative/hypnotic that increases cerebral blood flow (CBF), and its active metabolite 2,2,2-trichloroethanol (TCE) is an agonist for the nonclassical two-pore domain K(+) (K(2P)) channels TREK-1 and TRAAK. We sought to determine whether TCE dilates cerebral arteries in vitro by activating nonclassical K(+) channels. TCE dilated pressurized and perfused rat middle cerebral arteries (MCAs) in a manner consistent with activation of nonclassical K(+) channels. Dilation to TCE was inhibited by elevated external K(+) but not by an inhibitory cocktail (IC) of classical K(+) channel blockers. Patch-clamp electrophysiology revealed that, in the presence of the IC, TCE increased whole-cell currents and hyperpolarized the membrane potential of isolated MCA smooth muscle cells. Heating increased TCE-sensitive currents, indicating that the activated channel was thermosensitive. Immunofluorescence in sections of the rat MCA demonstrated that, like TREK-1, TRAAK is expressed in the smooth muscle of cerebral arteries. Isoflurane did not, however, dilate the MCA, suggesting that TREK-1 was not functional. These data indicate that TCE activated a nonclassical K(+) channel with the characteristics of TRAAK in rat MCA smooth-muscle cells. Stimulation of K(+) channels such as TRAAK in cerebral arteries may therefore explain in part how CH/TCE increases CBF.

Fig. 1.

Dilation of the rat MCA to TCE. Top, raw traces of arterial diameter. Bottom, summary data. A, in the control condition (●), denuded, pressurized, and perfused rat MCAs dilated in response to increasing concentrations of TCE (n = 5). B, replacing the control Krebs' buffer with a buffer containing isotonic 60 mM KCl (■) impaired the response to TCE (n = 8). C, inclusion of an inhibitory cocktail of classical K⁺ channel blockers (○) did not alter the dilation caused by TCE (n = 4). Summary data are means ± S.E.M.

Fig. 2.

TRAAK protein expression. A, Western blot showing TRAAK immunopositive bands at 43 kDa in rat cardiovascular tissues. Cerebral arteries = MCA+basilar. Positive (+) control is IMR-32 human neuroblastoma cell lysate (sc-2409, Santa Cruz Biotechnology). B, sections of rat brain and MCA imaged at three levels of magnification (10×, 40×, and 60× oil immersion) using epifluorescence microscopy. Tissue slices (8 μm) were incubated with a primary antibody directed toward TRAAK (right) or an equal amount of a control nonimmune IgG (left). Red fluorescence is TRAAK; blue is DAPI staining of nuclei.

Fig. 3.

Electrophysiology of isolated rat MCA smooth muscle cells. Whole-cell patch-clamp current recordings of freshly dissociated rat MCA smooth muscle cells. In response to a voltage-step protocol (signal, −80 to 80 mV, 60-ms pulses separated by 10 mV), TCE elicited concentration-dependent increases in current (mostly in the outward direction) under control conditions (left) and after inhibition of classical K⁺ channels with the IC (right). Note the difference in scale: Control is −400 to 3000 pA; inhibitory cocktail is −400 to 500 pA.

Fig. 4.

Summary data of currents activated by TCE in rat MCA smooth muscle cells. A to C, control cells (n = 5 animals) are on the left, and cells treated with the IC of classical K⁺ channel blockers (n = 5 animals) are on the right. A, at 10⁻² M, TCE increased peak outward current in both control cells and cells treated with the IC. In B and C, ●, I–V curves of baseline currents are shown; ○, currents stimulated by TCE at 10⁻³ M; ■, 10⁻² M TCE. B, under control conditions, TCE increased whole-cell currents at both 10⁻³ and 10⁻² M. Inclusion of the IC reduced overall whole-cell currents, and increases over baseline were only seen to 10⁻² M TCE. C, subtracting out the baseline responses generated difference currents representing the currents due only to TCE (I_TCE). Although the I_TCE was nonzero for both concentrations of TCE under control conditions, I_TCE at 10⁻² M was greater than at 10⁻³ M. In the presence of the IC, however, only 10⁻² M TCE was sufficient to generate currents that were nonzero, or different from baseline. Data are means ± S.E.M. *, statistically significant difference (p ≤ 0.05).

Fig. 5.

Changes in reversal potential with increased extracellular potassium. Top, traces of an rat MCA smooth muscle cell treated with the IC of classical K⁺ channel blockers and 10⁻² M TCE as the extracellular K⁺ concentration is raised from 5.4 mM to 54 and 140 mM. Bottom, summary of experimental data collected from rat MCA smooth muscle cells (red circles, n = 4 animals), and simulated pure K⁺ currents (black squares). Regression lines were fitted to the experimental (red) and simulated (blue) data to determine the slope of the response. Solid lines, best-fit line; dashes, 95% confidence interval of the best-fit line. Data are means± S.E.M. *, statistically significant difference (p ≤ 0.05).

Fig. 6.

Baseline response of rat MCA smooth muscles cell to heat. Whole-cell current traces of a MCA smooth muscle cell responding to a voltage-ramp protocol (stimulus, −100 to +100 mV in 700 ms) in the presence of the IC to block classical K⁺ channels. Heating the preparation from room temperature (22°C) to 33°C did not alter the baseline response of the cell.

Fig. 7.

Effect of heat on the TCE response in rat MCA smooth muscle cells. A, an example of a whole-cell voltage-clamp recording of an isolated rat MCA smooth muscle cell treated with 10⁻³ M TCE in the presence of the IC to block classical K⁺ channels. In response to a voltage-step protocol (inset) currents at 33°C were augmented over those at room temperature (22°C). B, summary data (n = 5 animals) showing that I–V curves of MCA smooth muscles cells at room temperature (22°C) at baseline (●), and after addition of 10⁻³ M TCE (○) were similar, but that simply heating to 33°C (■) enhanced the currents. C, summary of peak outward currents at room temperature and at 33°C in the presence of the IC and 10⁻³ M TCE. D, subtracting out the baseline I–V response revealed that the currents attributable to 10⁻³ M TCE (I_{TCE (-3)}) at 33°C were greater than zero, meaning that they were different from the baseline currents. E, I–V plot of difference currents due to 10⁻² M TCE at room temperature (I_{TCE (-2) RT}, ○) and to 10⁻³ M TCE at 33°C (I_{TCE (-3) 33°}, ●). Data are means ± S.E.M. *, statistically significant difference (p ≤ 0.05).