Halothane and Isoflurane Decrease the Open State Probability of Potassium sup + Channels in Dog Cerebral Arterial Muscle Cells 

These experiments were approved by the Medical College of Wisconsin Animal Care and Use Committee.

Adult mongrel dogs of either sex weighing 15–25 kg were placed in a plexiglass box and anesthetized with halothane. After attainment of surgical anesthesia, brains were removed. The middle cerebral arteries were dissected free of arachnoid and placed in cold Krebs' solution. Vessels were cut into small pieces and placed in 2-ml vials containing enzyme solution of the following composition (mM): NaCl 135, KCl 5.2, MgCl²1.0, HEPES 10, CaCl²0.04, glucose 10, and collagenase CLS II 500 U/ml (Worthington Biochemical, Freehold, NJ), dithiothreitol 4 mM (Sigma Chemical, St. Louis, MO) and papain 2 U/ml (Worthington Biochemical, Freehold, NJ). The enzyme solution was maintained at 37 degrees Celsius and stirred at 10 rpm by a microstirrer for 1–1.5 h. The supernatant was then removed and diluted with Tyrode's solution of the following composition (mM): NaCl 135, KCl 5.2, MgCl²1.0. HEPES 10, CaCl²1.8, and glucose 10. The dispersed cells were kept at 4 degrees Celsius until used.

Dispersed cells were placed in a perfusion chamber (22 degrees Celsius) on the stage of an inverted microscope (IMT-2, Olympus Optical, Tokyo, Japan) equipped with modulation contrast. At 500x magnification, a hydraulic micromanipulator (MO-203, Narishige, Tokyo, Japan) was used to position heat-polished borosilicate patch pipettes with tip resistance of 1–5 M Omega on the membranes of single cerebral arterial muscle cells. Once high resistance seals (2–10 G Omega) were formed, the pipette patch was removed by negative pressure to give access to the whole cell. Whole-cell Potassium sup + currents were elicited every 5–10 s by 200 ms depolarizing pulses generated by a computerized system (PClamp Software, Axon Instruments, Burlingame, CA). The currents were amplified by a List EPC-7 patch-clamp amplifier (List-Electronics, Darmstadt-Eberstadt, Germany), and the amplifier output was low-pass filtered at 1 KHz. All data were digitized (sampling rate = 10,000/s) and stored on a hard disk to permit analysis at a later time. Leak and capacitive currents in whole-cell recording were subtracted from each record by linearly summating scaled currents obtained during 10 mV hyperpolarizing pulses.

The composition of the pipette solution used for whole-cell Potassium sup + current recording was (in mM): KCl 70, Potassium sup +-glutamate 60, Potassium²ATP 5, EGTA 2.5, HEPES 10, MgCl²1, and CaCl²1.8; the computer-calculated free intracellular [Calcium sup 2+], using the program Chelator by Theo J. M. Shoenmakers, was 10 sup -6 M (pH = 7.2). A variant of this solution contained 10 mM EGTA and zero CaCl², giving a free [Calcium²+]ⁱof less or equal to 10 sup -9 M. The external solution contained (in mM): CaCl²1.8 NaCl 135, KCl 5.2, MgCl², 1.0, HEPES 10, and glucose 10 [pH = 7.4].

Single channel currents were recorded in a cell-attached configuration using the standard patch-clamp technique. [13] The bath solution was the same as that used for whole-cell recordings. The pipette solution contained (mM): KCl 145, CaCl²1.8, MgCl²1.1, and HEPES 5 (pH = 7.4).

Statistical analysis of single channel activity was performed using Pstat software (Axon, pClamp version 5.5). The mean open time, channel open probability (Po), and amplitude were analyzed for each cell and stored. Transitions from closed to open states were defined as a change in 50% below or above baseline relative to the predominant channel amplitude. The open state probability (NPo) was defined by the relation, NPo = I/i, where I is the time-averaged mean current, N is number of channels, i is single-channel current amplitude, and Po is channel open probability. Current-voltage relationships were fitted by least-square linear regression for determination of slope conductance.

After recording of Potassium sup + currents in control solutions, the inflow perfusate was changed to one in which a given concentration of anesthetic agent had been equilibrated at room temperature. Halothane was prepared in final bath concentrations of 0.4 and 0.9 mM, which was equivalent to 0.7 and 1.5%, respectively. [21] Isoflurane was prepared in a final bath concentration of 1.2 mM, which was equivalent to 2.7%. Anesthetic content of the perfusate from the perfusion chamber was sampled and verified by gas chromatography. Drug effects were completed within 2–3 min, and measurements were made after stabilization of drug-induced changes. The inflow perfusate containing the anesthetics was then changed to the control solution, and Potassium sup + channel currents were measured again.

Rings of middle cerebral arteries were mounted between two Tungsten wire triangles in 15 ml water-jacketed organ bath. Isometric tension was recorded on a model 7 Grass polygraph (Quincy, MA). The temperature was maintained at 37 degrees Celsius. The rings were stretched progressively to a final optimal tension of approximately 750 mg. The optimal tension was previously determined by length-tension studies using 40 mM KCl (unpublished observation). The integrity of each ring was examined by the contractile response to 40 mM KCl added to the bathing media. Isometric contractions were determined in middle cerebral arterial rings that were equilibrated in physiologic salt solution (pH 7.4) of the following composition (in mM): NaCl 119, KCl 4.7, CaCl²1.6, MgSO⁴1.17, NaHCO³27.8, NaH²PO⁴1.18, EDTA 0.026, glucose 5.5, and HEPES (4 (2-) 2-hydroxyethyl)(-1-piperazineethane-sulfonic acid) 5. The solution was aerated with 93.5% Oxygen²and 6.5% CO².

The arterial rings were washed repeatedly, and after a 60-min equilibration period, they were contracted with PGF²alpha (3 micro Meter) before and after 15 min exposure to tetraethylammonium. During a stable PGF²alpha-induced contraction, different concentrations of halothane or isoflurane were bubbled into the tissue bath using a vaporizer (Dragerwerk, Lubeck, Germany) to determine a dose-response relation. The preparations were exposed to volatile anesthetics for 15 min. Arterial rings were rinsed and rested for 30 min between exposures to different anesthetics. The concentrations of halothane (0.29 mM and 0.43 mM equivalent to 0.98% and 1.5%, respectively, at 37 degrees Celsius) and isoflurane (0.21 mM and 0.43 mM equivalent to 1.1% and 2.1%, respectively, at 37 degrees Celsius) in the bath were determined by gas chromatography.

Results are expressed as mean plus/minus SEM. Where appropriate, data were analyzed by Student's t test (P < 0.05).

(Figure 1(A)) depicts a macroscopic outward current obtained from single canine middle cerebral arterial smooth muscle cell dialyzed with a pipette solution containing a free intracellular calcium concentration of 1 micro Meter to enhance Calcium²+-dependent Potassium sup + current. The current was activated progressively by 200 ms depolarizing pulses from a holding potential of -60 mV (Figure 1(A), top) to consecutive more positive membrane potentials. Stepwise depolarization from a holding potential of -60 mV to beyond -30 mV elicited an outward current, which showed a mean peak amplitude of 1041 plus/minus 140 pA at +60 mV (n = 10). We examined the effect of 3 mM tetraethylammonium (TEA), a Calcium²+-activated Potassium sup + channel blocker, [11–14] on this outward current. As illustrated in Figure 1(B), addition of TEA significantly decreased peak current amplitude at +60 mV by 65 plus/minus 5%.

The effects of halothane on this TEA-sensitive Potassium sup + current was examined in eight cells. Representative tracings showing the effects of low and high doses of halothane on Potassium sup + currents elicited by depolarizing pulses (200 ms duration) from -60 mV to +60 mV are presented in Figure 2(top). Halothane reversibly and dose-dependently suppressed macroscopic Potassium sup + channel current. The effects of halothane on the current-voltage (I-V) relationship for Potassium sup + channel activation are shown in Figure 2(bottom). This anesthetic agent produced concentration-dependent suppression of the Potassium sup + channel current amplitude over the entire voltage range studied, without shifting the voltage-dependency of the I-V relationship. Low and high concentrations of halothane depressed peak Potassium sup + current at +60 mV by 18 plus/minus 4% and 34% plus/minus 6%, respectively.

In a separate series of experiments, we used a whole-cell mode in which [Calcium²+]ⁱwas strongly buffered with 10 mM-EGTA in the pipette solution [11] to minimize opening of the Calcium²+-dependent Potassium sup + channel current and examined the effect of halothane on Calcium²+-independent Potassium sup + channel current. Figure 3(A) depicts a macroscopic outward current recorded from canine middle cerebral arterial muscle cell and represents the most commonly encountered current pattern. The current was activated progressively by 200 ms depolarizing pulses from a holding potential of -60 mV (Figure 3(A), top) to consecutive more positive membrane potentials. The mean peak current in these cells was 423 plus/minus 58 pA at +60 mV (n = 7). This outward current displayed properties of a delayed rectifier Potassium sup + current, that is the rate of activation became faster at more positive voltages and exhibited very little inactivation during 200 ms command pulses. Application of 3 mM TEA had little effect on this outward current (n = 4). Addition of 1 micro Meter Calcium²+ ionophore (A23187) also did not affect the amplitude of this outward Potassium sup + current (n = 3), indicating that the high EGTA concentration (10 mM) in the pipette solution strongly buffered changes in [Calcium²+]ⁱto minimize the Calcium²+-activated Potassium sup + current in these cells. We then examined the effects of 4-aminopyridine (1 mM, 4-AP), a Potassium sup + channel blocker, on this outward current. Recordings in Figure 3(B) show that 4-AP significantly decreased the peak amplitude of this current. Figure 3(C) summarizes the current-voltage (I-V) relationship plotted as percent of maximum current before and after the addition of 4-AP to the external solution in four paired experiments. Application of 1 mM 4-AP reduced the peak current at +60 mV by 51 plus/minus 4%. When Potassium sup + glutamate and KCl were replaced with CsCl in the pipette solution, we were unable to measure an outward current (n = 4). suggesting that Potassium sup + was the charge carrier.

We studied the effects of halothane on this Calcium²+-independent Potassium sup + current in 12 cells. Representative tracings showing the effects of low and high doses of halothane on the Calcium²+-independent Potassium sup + channel currents, elicited by depolarizing pulses (200 ms duration) from -60 mV to +60 mV, are presented in Figure 4(top). Halothane reversibly and dose-dependently suppressed macroscopic Potassium sup + channel current. The effect of halothane on the current-voltage (I-V) relationship for Potassium sup + channel activation is shown in Figure 4(bottom). Low and high doses of this anesthetic agent produced concentration-dependent suppression of Potassium sup + channel current amplitude over the entire voltage range studied, without shifting the voltage dependency of the I-V relationship. Low and high concentrations of halothane depressed peak Potassium sup + current at +60 mV by 17 plus/minus 5% and 29 plus/minus 7%, respectively. However, there was no significant difference in the sensitivity of Calcium²+-dependent and -independent Potassium sup + currents to halothane.

After macroscopic current measurements, the effects of halothane also were examined on single-channel Potassium sup + currents recorded from cell-attached patches of dog middle cerebral arterial muscle cells. We also analyzed the effect of isoflurane on single-channel Potassium sup + currents, because our laboratories have shown earlier that this anesthetic agent also decreases whole-cell Potassium sup + current in cerebral arterial muscle cells. [22]Figure 5(A) shows representative tracings of single-channel Potassium sup + currents recorded at various pipette potentials from cell-attached patches. The current-voltage relationship of this single-channel Potassium sup + current recorded from six cells revealed a unitary slope conductance of 99 pS when determined at pipette potentials between +40 mV and -40 mV (Figure 5(B)). Assuming that intracellular Potassium sup + concentration was comparable to that in the pipette solution, the absence of detectable Potassium sup + currents at a pipette potential of -60 mV suggested that intracellular membrane potential may reflect this value. When incremental negative pipette potentials were applied, there was reversal of single-channel current at potentials negative to -60 mV (Figure 5). Application of tetraethylammonium (TEA, 0.1–3.0 mM) to the extracellular membrane surface via the pipette solution, produced a concentration-dependent reduction of the single-channel current amplitude (Figure 6). Figure 7illustrates representative tracings showing the effect of the Calcium²+ ionophore (A23187) on single channel Potassium sup + currents. Addition of the Calcium²+ ionophore (A23187, 1 micro Meter) in the bathing solution increased the open state probability of the channel from 0.015 plus/minus 0.001 to 0.040 plus/minus 0.021 (p < 0.05) with out a significant reduction on unitary current amplitude. These findings suggest that the 99 pS Potassium sup + channel is a Calcium²+-activated Potassium sup + channel. In some patches, a smaller amplitude current also was observed, indicating the presence of second channel type or a subconductance state of the 99 pS Potassium sup + channel (Figure 8). These smaller openings were observed in only 10% of patches. However, the detection of this channel may have been influenced by the predominance of the 99 pS channel type, which made single-channel analysis of the small amplitude current difficult.

The effects of halothane and isoflurane on these 99 pS Potassium sup + currents were determined in cell-attached patches of dog middle cerebral arterial muscle cells. Figure 9depicts the summary of statistical analysis of mean open time, open state probability, and event frequency obtained before, during, and after exposure to 0.9 mM halothane. Halothane decreased event frequency from 28.1 plus/minus 6.9 to 14.9 plus/minus 3.4 per 2-min recording interval, the mean open time from 14.1 plus/minus 1.3 ms to 9.4 plus/minus 1.5 ms (Figure 9; n = 9, P < 0.05), and the open state probability (NP⁰) of the 99 pS Potassium sup + channels from 0.011 plus/minus 0.005 to 0.004 plus/minus 0.002. Figure 10shows representative tracings obtained before, during, and after exposure to 0.9 mM halothane at a pipette potential of -20 mV. The number of openings was greatly reduced during exposure to halothane. This effect of halothane on single-channel was reversed when the inflow perfusate was changed to the control solution. Halothane (0.9 mM) did not affect single channel amplitude, which was -3.0 plus/minus 0.2 pA and -2.9 plus/minus 0.2 pA at a pipette potential of -20 mV in the absence and presence of halothane, respectively (n = 9). This anesthetic had no significant effect on the slope of the I-V relationship, which was 99 pS in the absence and 93 pS in its presence. Similarly, the distribution of the unitary current amplitude was not altered, suggesting that halothane did not influence a separate conductance pathway but rather decreased the activity of the same channel type.

Similar experiments were performed at a single-channel level to examine the effects of isoflurane (1.2 mM) on the 99 pS Potassium sup + channel. Figure 11depicts single-channel recordings obtained before, during, and after exposure to 1.2 mM isoflurane. Single channel currents were recorded at a pipette potential of -20 mV. Similar to halothane, the number of single channel events seen during exposure to isoflurane was reversibly reduced; also like halothane, isoflurane did not affect single channel amplitude which was -3.4 plus/minus 0.2 pA and -3.3 plus/minus 0.2 pA at a pipette potential of -20 mV in the absence and in the presence of isoflurane, respectively (n = 5). Isoflurane decreased event frequency from 21.0 plus/minus 3.6 to 10.7 plus/minus 3.7 per 2-min recording interval, and the mean open time from 19.1 plus/minus 2.7 ms to 13.0 plus/minus 1.5 ms. The open state probability (NP⁰) was decreased from 0.0109 plus/minus 0.0025 to 0.0053 plus/minus 0.0019 in the presence of isoflurane.

In an attempt to examine whether blockade of the Calcium²+-activated Potassium sup + channel interferes with anesthetic-induced vasodilation of middle cerebral arteries, the effects of halothane and isoflurane was determined before and after treatment of the vessels with TEA. The vasodilator action of halothane and isoflurane on middle cerebral arterial rings (number of vessels = 28, number of animals = 4) preconstricted with PGF²alpha (3 micro Meter) in the absence and presence of TEA are summarized in Table 1. Treatment of the vessels with 1 or 3 mM TEA, a blocker of the Calcium²+-activated Potassium sup + channel, [11,13] produced no significant effect on halothane- or isoflurane-induced vasodilation in canine cerebral arterial vessels. These data suggest that the anesthetic-induced vasodilation in cerebral arterial smooth muscle does not involve Calcium²+-dependent Potassium sup + channels.

In the current study, using 2.5 mM EGTA and 1.8 mM CaCl²(intracellular [Calcium²+] of 1 micro Meter) in the pipette solution, we have recorded a macroscopic outward Potassium sup + current that was suppressed by 3 mM TEA in dog cerebral arterial muscle cells. The current did not inactivate significantly over a period of 200 ms. The rate of activation of this current became faster at more positive potentials demonstrating voltage-dependence of activation. A similar TEA-sensitive Calcium²+-activated macroscopic Potassium sup + current has been described in number of vascular smooth muscle cells including rabbit portal veins [12,18] and rat mesenteric arteries. [11] In dog cerebral arterial muscle cells dialyzed with pipette solution containing zero Calcium²+ and 10 mM EGTA to buffer the intracellular Calcium²+([Calcium²+]ⁱof less or equal to 1 nM), we also recorded a macroscopic outward Potassium sup + current that was TEA (3 mM)-insensitive but 4-aminopyridine sensitive, thus displaying properties of a delayed rectifier Potassium sup + current. A similar outward, delayed rectifier Potassium sup + current has ben described in cat cerebral [16] and rabbit pulmonary arterial muscle cells. [17].

The inhalational anesthetic halothane similarly depressed these two Potassium sup + current types in canine cerebral arterial cells. Unlike these results, recordings in bovine adrenal chromaffin cells have shown that inhalational anesthetics have a pronounced effect on Calcium²+-dependent macroscopic Potassium sup + current as compared to Calcium²+-independent Potassium sup + current. [23] These discrepancies might be due to tissue differences as well as methodologic differences, because [Calcium²+]ⁱwas fixed in the current study but not in the bovine adrenal chromaffin cell study. In the latter study, blockade of Calcium²+ influx by anesthetics may have contributed to the more pronounced depression of Calcium²+-sensitive Potassium sup + channel current. [23].

In the current study, using 145 mM KCl in the patch pipette and 5.2 mM KCl in the bath, we recorded a predominant potassium channel type with a unitary conductance of 99 pS from dog cerebral arterial muscle in the cell-attached mode. In some patches, a smaller amplitude current, which also could be a subconductance state of the 99 pS channel, was observed but not analyzed. The 99 pS Potassium sup + channel was voltage-sensitive and blocked by tetraethylammonium, and its open state probability (NPo) was enhanced by external application of Calcium²+ ionophore A23187 (1 micro Meter), indicating Calcium²+ sensitivity. A23187, at the concentration used in the present study, produced statistically insignificant reduction in unitary amplitude of the Calcium²+-activated Potassium sup + current in cell-attached patches, which may suggest a lack of effect on the cell membrane potential. The properties of this large conductance Potassium sup + channel are consistent with the Calcium²+-activated “maxi Potassium sup +” channel described in a number of smooth muscle cell types. [11,14].

The major findings of the current study is that halothane and isoflurane, at clinically relevant concentrations, reduced the open state probability of the predominant Calcium²+-activated Potassium sup + channel without changing its single-channel conductance. This decrease in open state probability appears to have resulted from a decrease in both the frequency of channel opening and the mean open time. Although the present study is the first to show the effect of halothane and isoflurane on the activity of single channel Potassium sup + current in vascular muscle membranes, the effects of anesthetics on Calcium²+-activated Potassium sup + channel with similar conductance properties have been reported on rat glioma C6 cells. [21] Chara australis, [25] and bovine adrenal chromaffin cells. [23,26] In rat glioma C6 cells, volatile anesthetics, including halothane and isoflurane, reduced rubidium efflux across Calcium²+-activated Potassium sup + channels. [24] In bovine adrenal chromaffin cells, both halothane and enflurane reduced mean open time without affecting the single channel conductance of Calcium²+-activated Potassium sup 1 channel, [23,26] which is in agreement with our current observations in canine cerebral arterial smooth muscle cells.

The results of the current studies also indicate that halothane and isoflurane did not enhance Potassium sup + current, which would be expected to promote vasodilation but instead reduced the activity of Potassium sup + channels in cerebral arterial muscle membranes. Recent studies from our laboratories also have shown that isoflurane reduces whole-cell Potassium sup + current in canine cerebral arterial cells. [22] These results suggest that mechanisms other than Potassium sup + channel opening likely mediate volatile anesthetic-induced vasodilation. Furthermore, in isometric tension recording studies, blockade of Calcium²+ activated Potassium sup + current with 1 or 3 mM TEA produced no significant effect on halothane- or isoflurane-induced vasodilation. However, recent studies from our laboratories and others have shown that volatile anesthetics reduce whole-cell Calcium²+ current in cardiac [8,9] as well as in vascular smooth muscle cells. [10] This suppression of Calcium²+ current may be one of several mechanisms by which halothane and isoflurane relax cerebral arterial muscle. Halothane also has been reported to decrease Calcium²+ accumulation in the sarcoplasmic reticulum and attenuate the Calcium²+ activation of contractile proteins in rabbit aortic strips, [5] thus resulting in less Calcium sup 2+ contractile protein interaction, which is responsible for arterial contraction. Halothane also was reported to increase tissue cGMP levels in mouse heart [27] and canine cerebral arteries, [28] which might be an alternative mechanism for anesthetic-induced vasodilation, as an increase in tissue cGMP level is associated with vascular muscle relaxation. [29].

Although our findings did not identify the site of action of halothane and isoflurane, they provide relevant evidence for the possible mode of action of these volatile anesthetics at cellular level. In the current study, both isoflurane and halothane decreased event frequency and mean open time of a Calcium²+-activated Potassium sup + channel. One interpretation of these observations is that these anesthetic agents may stabilize the Potassium sup + channel in the resting as well as in the inactivated state. [30] In the presence of 0.9 mM halothane or 1.2 mM isoflurane, some of the Potassium sup + channels may remain in their resting state, and these channels would activate normally and exhibit normal open-channel Potassium sup + conduction. However, after opening, the channel may go into the inactivated conformation fast or close more rapidly, leading to a net reduction in the open state duration.

In conclusion, halothane suppresses the macroscopic Potassium sup + current, both the Calcium²+-independent as well as the Calcium²+-sensitive Potassium sup + channel current, in cerebral arterial muscle cells. At the single channel level, halothane, like isoflurane, also reduces the mean open time, event frequency, and open state probability of Calcium²+-sensitive Potassium sup + channel current in cerebral arterial muscle cells. In cerebral arterial segments, block of Calcium²+-sensitive Potassium sup + channel by TEA produced no significant effect on volatile anesthetic-induced vasodilation. Thus, these findings suggest that halothane- and isoflurane-induced vasodilations are not mediated through modulation of Potassium sup + channels in canine cerebral arterial cells.

The authors thank Mimi Mick, for her secretarial assistance, and Dr. David R. Harder, for allowing the single channel experiments to be conducted in his laboratory.