Free
Meeting Abstracts  |   February 1995
Halothane and Isoflurane Decrease the Open State Probability of Potassium sup + Channels in Dog Cerebral Arterial Muscle Cells 
Author Notes
  • (Eskinder) Assistant Professor, Department of Anesthesiology.
  • (Gebremedhin) Assistant Professor, Department of Physiology.
  • (Lee) Visiting Professor, Department of Anesthesiology.
  • (Rusch) Associate Professor, Department of Physiology.
  • (Supan) Research associate, Department of Anesthesiology.
  • (Kampine) Professor and Chairman, Department of Anesthesiology; Professor of Physiology
  • (Bosnjak) Professor, Departments of Anesthesiology and Physiology.
  • Received from the Departments of Anesthesiology and Physiology. The Medical College of Wisconsin, and the Zablocki Veterans Affairs Medical Center, Milwaukee, Wisconsin. Submitted for publication August 20, 1991. Accepted for publication October 6, 1994.
  • Address correspondence to Dr. Eskinder: Department of Anesthesiology, Medical College of Wisconsin, MEB 162C.8701 Watertown Plank Road, Milwaukee, Wisconsin 53226.
Article Information
Meeting Abstracts   |   February 1995
Halothane and Isoflurane Decrease the Open State Probability of Potassium sup + Channels in Dog Cerebral Arterial Muscle Cells 
Anesthesiology 2 1995, Vol.82, 479-490. doi:
Anesthesiology 2 1995, Vol.82, 479-490. doi:
Key words: Anesthetics, volatile: halothane; isoflurane, Circulation, cerebral: middle cerebral artery, Current: Potassium sup +. Patch-clamps: single channel.
THE volatile anesthetic halothane is a potent cerebral vasodilator. It increases cerebral blood flow in a concentration-dependent manner both in animals [1 ] and humans. [2 ] Because isoflurane causes a relatively less cerebral vasodilation than does halothane, [3 ] it is the anesthetic of choice for neurosurgical procedures. Both isoflurane and halothane also relax other vascular beds including isolated rat [4 ] and rabbit aorta, [5 ] canine carotid artery, [6 ] and porcine coronary artery. [7 ] Although volatile anesthetics are potent vasodilators, the mechanisms by which they relax vascular smooth muscle are not well understood. Recent studies from our laboratory have shown that volatile anesthetics suppress voltage-dependent Calcium2+ channel current in both cardiac [8,9 ] and vascular smooth muscle cells. [10 ] These findings indicate that one possible mechanism by which volatile anesthetics dilate vascular smooth muscle might be via a direct effect on Calcium2+ channel currents.
However, one other potential mechanism for arterial vasodilation is enhanced Potassium sup + efflux, enabled by an increased open state probability of membrane Potassium sup + channels. Based on pharmacologic and biophysical properties, a number of Potassium sup + channel types have been identified in vascular smooth muscle membranes. [11–18 ] The most common Potassium sup + channel in vascular smooth muscle is the Calcium2+-activated Potassium sup + channel. The open state probability of this channel is low at resting membrane potential but increases in proportion to membrane depolarization and elevation of intracellular Calcium2+ concentration. [11–13 ] The resulting enhanced Potassium sup + efflux will induce membrane repolarization or hyperpolarization, [11–15 ] reduce the open state probability of voltage-dependent Calcium2+ channels, and in turn cause vascular relaxation. In addition to this Potassium sup + channel type, an outwardly rectifying (delayed rectifier) Potassium sup + current that is voltage- but not Calcium2+-dependent has been observed in vascular muscle membranes from cat cerebral artery, [16 ] rabbit portal vein. [18 ] An inward rectifier Potassium sup + current activated at potentials close to the Potassium sup + equilibrium potential also has been reported in vascular smooth muscle. [19 ] This Potassium sup + channel type may play a role in setting the level of the resting membrane potential. [19 ] Another distinct Potassium sup + channel in vascular smooth muscle cells is the ATP-sensitive (Potassium sub ATP) channel that is activated by low cytosolic ATP levels. [20 ] The PotassiumATPchannel is only weakly voltage-sensitive and is not activated by changes in intracellular Calcium2+ concentrations.
Even though a substantial number of Potassium sup + channel types are found in vascular smooth muscle cells, the direct effect of volatile anesthetics on these Potassium sup + channels in arterial membranes is not well understood. Therefore, in the current study we applied the patch-clamp technique to examine the effect of halothane and isoflurane on Potassium sup + channel currents in isolated dog cerebral arterial muscle cell membranes.
Methods
These experiments were approved by the Medical College of Wisconsin Animal Care and Use Committee.
Cell Isolation
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.
Voltage-Clamp Recording
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.
Isometric Tension Recording
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.
Statistics
Results are expressed as mean plus/minus SEM. Where appropriate, data were analyzed by Student's t test (P < 0.05).
Results
Effect of Halothane on Macroscopic Potassium sup + Current
(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%.
Figure 1. Whole-cell outward Potassium sup + current in a single canine cerebral arterial muscle cell dialized with 1.8 mM CaCl2and 2.5 mM EGTA before (A) and after (B) addition of 3 mM tetraethylammonium (TEA). Cells were progressively depolarized from a holding potential of 60 mV to the corresponding potentials indicated on the pulse protocol. An outward current was elicited whose amplitude was markedly depressed by TEA. (C) Peak current-voltage relations obtained before and after exposure to 3 mM TEA in two canine cerebral arterial muscle cells.
Figure 1. Whole-cell outward Potassium sup + current in a single canine cerebral arterial muscle cell dialized with 1.8 mM CaCl2and 2.5 mM EGTA before (A) and after (B) addition of 3 mM tetraethylammonium (TEA). Cells were progressively depolarized from a holding potential of 60 mV to the corresponding potentials indicated on the pulse protocol. An outward current was elicited whose amplitude was markedly depressed by TEA. (C) Peak current-voltage relations obtained before and after exposure to 3 mM TEA in two canine cerebral arterial muscle cells.
Figure 1. Whole-cell outward Potassium sup + current in a single canine cerebral arterial muscle cell dialized with 1.8 mM CaCl2and 2.5 mM EGTA before (A) and after (B) addition of 3 mM tetraethylammonium (TEA). Cells were progressively depolarized from a holding potential of 60 mV to the corresponding potentials indicated on the pulse protocol. An outward current was elicited whose amplitude was markedly depressed by TEA. (C) Peak current-voltage relations obtained before and after exposure to 3 mM TEA in two canine cerebral arterial muscle cells.
×
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.
Figure 2. Modulation of macroscopic Potassium sup + channel current by low (0.4 mM equivalent to 0.7%) and high (0.9 mM, equivalent to 1.5%) concentrations of halothane in canine cerebral arterial muscle cells dialized with 1.8 mM CaCl and 2.5 mM EGTA. (Top) The pulse protocol and actual recordings of macroscopic Potassium sup + current traces in control solution and after exposure to low or high doses of halothane. Voltage steps from a holding potential (HP) of 60 mV to the corresponding potentials elicited an outward current whose amplitude was depressed markedly and reversibly by halothane. (Bottom) The peak current-voltage relationship in control solution and after exposure to low and high doses of halothane, 0.4 (n = 4) and 0.9 mM (n = 5), respectively. Cells were depolarized progressively from a HP of 60 mV to the corresponding potentials indicated on the abscissa. Points with error bars represent the mean plus/minus SEM.
Figure 2. Modulation of macroscopic Potassium sup + channel current by low (0.4 mM equivalent to 0.7%) and high (0.9 mM, equivalent to 1.5%) concentrations of halothane in canine cerebral arterial muscle cells dialized with 1.8 mM CaCl and 2.5 mM EGTA. (Top) The pulse protocol and actual recordings of macroscopic Potassium sup + current traces in control solution and after exposure to low or high doses of halothane. Voltage steps from a holding potential (HP) of 60 mV to the corresponding potentials elicited an outward current whose amplitude was depressed markedly and reversibly by halothane. (Bottom) The peak current-voltage relationship in control solution and after exposure to low and high doses of halothane, 0.4 (n = 4) and 0.9 mM (n = 5), respectively. Cells were depolarized progressively from a HP of 60 mV to the corresponding potentials indicated on the abscissa. Points with error bars represent the mean plus/minus SEM.
Figure 2. Modulation of macroscopic Potassium sup + channel current by low (0.4 mM equivalent to 0.7%) and high (0.9 mM, equivalent to 1.5%) concentrations of halothane in canine cerebral arterial muscle cells dialized with 1.8 mM CaCl and 2.5 mM EGTA. (Top) The pulse protocol and actual recordings of macroscopic Potassium sup + current traces in control solution and after exposure to low or high doses of halothane. Voltage steps from a holding potential (HP) of 60 mV to the corresponding potentials elicited an outward current whose amplitude was depressed markedly and reversibly by halothane. (Bottom) The peak current-voltage relationship in control solution and after exposure to low and high doses of halothane, 0.4 (n = 4) and 0.9 mM (n = 5), respectively. Cells were depolarized progressively from a HP of 60 mV to the corresponding potentials indicated on the abscissa. Points with error bars represent the mean plus/minus SEM.
×
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.
Figure 3. Whole-cell outward Potassium sup + current in a single canine cerebral arterial muscle cell dialized with zero Calcium2+ and 10 mM EGTA before (A) and after (B) addition of 1 mM 4-aminopyridine (4-AP). Cells were progressively depolarized from a holding potential of 60 mV to the corresponding potentials indicated on the pulse protocol. An outward current was elicited whose amplitude was markedly depressed by 4-AP. (C) Peak current-voltage relations obtained before and after exposure to 1 mM 4-AP in four canine cerebral arterial muscle cells. Points with error bars represent the mean plus/minus SEM.
Figure 3. Whole-cell outward Potassium sup + current in a single canine cerebral arterial muscle cell dialized with zero Calcium2+ and 10 mM EGTA before (A) and after (B) addition of 1 mM 4-aminopyridine (4-AP). Cells were progressively depolarized from a holding potential of 60 mV to the corresponding potentials indicated on the pulse protocol. An outward current was elicited whose amplitude was markedly depressed by 4-AP. (C) Peak current-voltage relations obtained before and after exposure to 1 mM 4-AP in four canine cerebral arterial muscle cells. Points with error bars represent the mean plus/minus SEM.
Figure 3. Whole-cell outward Potassium sup + current in a single canine cerebral arterial muscle cell dialized with zero Calcium2+ and 10 mM EGTA before (A) and after (B) addition of 1 mM 4-aminopyridine (4-AP). Cells were progressively depolarized from a holding potential of 60 mV to the corresponding potentials indicated on the pulse protocol. An outward current was elicited whose amplitude was markedly depressed by 4-AP. (C) Peak current-voltage relations obtained before and after exposure to 1 mM 4-AP in four canine cerebral arterial muscle cells. Points with error bars represent the mean plus/minus SEM.
×
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.
Figure 4. Modulation of macroscopic Potassium sup + channel current by low (0.4 mM equivalent to 0.7%) and high (0.9 mM, equivalent to 1.5%) concentrations of halothane in canine cerebral arterial muscle cells dialized with zero Calcium2+ and 10 mM EGTA. (Top) The pulse protocol and actual recordings of macroscopic Potassium sup + current traces in control solution and after exposure to low or high doses of halothane. Voltage steps from a holding potential (HP) of 60 mV to the corresponding potentials elicited an outward current whose amplitude was depressed markedly and reversibly by halothane. (Bottom) The peak current-voltage relationship in control solution and after exposure to low and high doses of halothane, 0.4 (n = 6) and 0.9 mM (n = 6), respectively. Cells were depolarized progressively from a HP of 60 mV to the corresponding potentials indicated on the abscissa. Points with error bars represent the mean plus/minus SEM.
Figure 4. Modulation of macroscopic Potassium sup + channel current by low (0.4 mM equivalent to 0.7%) and high (0.9 mM, equivalent to 1.5%) concentrations of halothane in canine cerebral arterial muscle cells dialized with zero Calcium2+ and 10 mM EGTA. (Top) The pulse protocol and actual recordings of macroscopic Potassium sup + current traces in control solution and after exposure to low or high doses of halothane. Voltage steps from a holding potential (HP) of 60 mV to the corresponding potentials elicited an outward current whose amplitude was depressed markedly and reversibly by halothane. (Bottom) The peak current-voltage relationship in control solution and after exposure to low and high doses of halothane, 0.4 (n = 6) and 0.9 mM (n = 6), respectively. Cells were depolarized progressively from a HP of 60 mV to the corresponding potentials indicated on the abscissa. Points with error bars represent the mean plus/minus SEM.
Figure 4. Modulation of macroscopic Potassium sup + channel current by low (0.4 mM equivalent to 0.7%) and high (0.9 mM, equivalent to 1.5%) concentrations of halothane in canine cerebral arterial muscle cells dialized with zero Calcium2+ and 10 mM EGTA. (Top) The pulse protocol and actual recordings of macroscopic Potassium sup + current traces in control solution and after exposure to low or high doses of halothane. Voltage steps from a holding potential (HP) of 60 mV to the corresponding potentials elicited an outward current whose amplitude was depressed markedly and reversibly by halothane. (Bottom) The peak current-voltage relationship in control solution and after exposure to low and high doses of halothane, 0.4 (n = 6) and 0.9 mM (n = 6), respectively. Cells were depolarized progressively from a HP of 60 mV to the corresponding potentials indicated on the abscissa. Points with error bars represent the mean plus/minus SEM.
×
Effect of Halothane and Isoflurane on Single-channel Potassium sup + Current
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.
Figure 5. Effect of changes in pipette potential on single-channel current amplitude in cell-attached patches. (A) Representative tracings of single-channel activity taken at various pipette potentials (PP) in patches from two different cerebral arterial muscle cells, c - closed state of the channel. (B) The current-voltage (I-V) relationship for this channel obtained from six arterial membrane patches. The slope of the curve determined via linear regression analysis was 99 pS. Each point represents the mean plus/minus SEM.
Figure 5. Effect of changes in pipette potential on single-channel current amplitude in cell-attached patches. (A) Representative tracings of single-channel activity taken at various pipette potentials (PP) in patches from two different cerebral arterial muscle cells, c - closed state of the channel. (B) The current-voltage (I-V) relationship for this channel obtained from six arterial membrane patches. The slope of the curve determined via linear regression analysis was 99 pS. Each point represents the mean plus/minus SEM.
Figure 5. Effect of changes in pipette potential on single-channel current amplitude in cell-attached patches. (A) Representative tracings of single-channel activity taken at various pipette potentials (PP) in patches from two different cerebral arterial muscle cells, c - closed state of the channel. (B) The current-voltage (I-V) relationship for this channel obtained from six arterial membrane patches. The slope of the curve determined via linear regression analysis was 99 pS. Each point represents the mean plus/minus SEM.
×
Figure 6. Representative tracings showing concentration-dependent reduction by tetraethylammonium (TEA) of single-channel amplitude at pipette potential (PP) of -20 mV, c - closed state of the channel.
Figure 6. Representative tracings showing concentration-dependent reduction by tetraethylammonium (TEA) of single-channel amplitude at pipette potential (PP) of -20 mV, c - closed state of the channel.
Figure 6. Representative tracings showing concentration-dependent reduction by tetraethylammonium (TEA) of single-channel amplitude at pipette potential (PP) of -20 mV, c - closed state of the channel.
×
Figure 7. Representative traces showing the effect of Calcium2+ ionophore (A23187, 1 micro Meter) on single-channel activity at pipette potential (PP) of 0 mV, c - closed state of the channel.
Figure 7. Representative traces showing the effect of Calcium2+ ionophore (A23187, 1 micro Meter) on single-channel activity at pipette potential (PP) of 0 mV, c - closed state of the channel.
Figure 7. Representative traces showing the effect of Calcium2+ ionophore (A23187, 1 micro Meter) on single-channel activity at pipette potential (PP) of 0 mV, c - closed state of the channel.
×
Figure 8. Two single-channel current amplitudes recorded at pipette potential (PP) of 20 mV in two cell-attached patches from two different canine cerebral arterial muscle cells. (A) Channel activity showing small amplitude channel. (B) Channel activity showing large amplitude channel. c closed state of the channel.
Figure 8. Two single-channel current amplitudes recorded at pipette potential (PP) of 20 mV in two cell-attached patches from two different canine cerebral arterial muscle cells. (A) Channel activity showing small amplitude channel. (B) Channel activity showing large amplitude channel. c closed state of the channel.
Figure 8. Two single-channel current amplitudes recorded at pipette potential (PP) of 20 mV in two cell-attached patches from two different canine cerebral arterial muscle cells. (A) Channel activity showing small amplitude channel. (B) Channel activity showing large amplitude channel. c closed state of the channel.
×
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.
Figure 9. Summary of statistical analysis of mean open time, open state probability (NPo), and event frequency (pipette potential -20 mV) single-channel Potassium sup + currents recorded from isolated canine middle cerebral arterial muscle cells before, during, and after halothane (0.9 mM, n - 9). All parameters were decreased significantly in the presence of halothane. This effect of halothane was reversed when the inflow perfusate was changed to the control solution. *Significant difference from control values at P < 0.05.
Figure 9. Summary of statistical analysis of mean open time, open state probability (NPo), and event frequency (pipette potential -20 mV) single-channel Potassium sup + currents recorded from isolated canine middle cerebral arterial muscle cells before, during, and after halothane (0.9 mM, n - 9). All parameters were decreased significantly in the presence of halothane. This effect of halothane was reversed when the inflow perfusate was changed to the control solution. *Significant difference from control values at P < 0.05.
Figure 9. Summary of statistical analysis of mean open time, open state probability (NPo), and event frequency (pipette potential -20 mV) single-channel Potassium sup + currents recorded from isolated canine middle cerebral arterial muscle cells before, during, and after halothane (0.9 mM, n - 9). All parameters were decreased significantly in the presence of halothane. This effect of halothane was reversed when the inflow perfusate was changed to the control solution. *Significant difference from control values at P < 0.05.
×
Figure 10. Representative traces of single-channel activity before, during, and after exposure to halothane (0.9 mM, equivalent to 1.5%) at pipette potential (PP) of 20 mV.c closed current state.
Figure 10. Representative traces of single-channel activity before, during, and after exposure to halothane (0.9 mM, equivalent to 1.5%) at pipette potential (PP) of 20 mV.c closed current state.
Figure 10. Representative traces of single-channel activity before, during, and after exposure to halothane (0.9 mM, equivalent to 1.5%) at pipette potential (PP) of 20 mV.c closed current state.
×
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.
Figure 11. Representative traces of single-channel activity before, during, and after exposure to isoflurane (1.2 mM, equivalent to 2.7%) at pipette potential (PP) of 20 mV.c - closed channel state.
Figure 11. Representative traces of single-channel activity before, during, and after exposure to isoflurane (1.2 mM, equivalent to 2.7%) at pipette potential (PP) of 20 mV.c - closed channel state.
Figure 11. Representative traces of single-channel activity before, during, and after exposure to isoflurane (1.2 mM, equivalent to 2.7%) at pipette potential (PP) of 20 mV.c - closed channel state.
×
Effect of Tetraethylammonium on Volatile Anesthetic-induced Vasodilation
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.
Table 1. Effect of TEA (1 and 3 mM) on 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)-Induced Vasodilation in Dog Cerebral Arterial Vessels
Image not available
Table 1. Effect of TEA (1 and 3 mM) on 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)-Induced Vasodilation in Dog Cerebral Arterial Vessels
×
Discussion
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.
REFERENCES
Christensen MS, Hoedt-Rasmussen K, Lassen NA: Cerebral vasodilation by halothane anaesthesia in man and its potentiation by hypotension and hypercapnia. Br J Anaesth 39:927-934, 1967.
Morita H, Nemoto EM, Bleyaert AL, Stezoski SW: Brain blood flow autoregulation and metabolism during halothane anesthesia in monkeys. Am J Physiol 233:H670-H676, 1977.
Drummond JC, Todd MM, Scheller MM, Shapiro HM: A comparison of the direct cerebral vasodilating potencies of halothane and isoflurane in the New Zealand white rabbit. ANESTHESIOLOGY 65:462-467, 1986.
Sprague DH, Yang JC, Ngai SH: Effects of isoflurane and halothane on contractility and cyclic 3′5′-adenosine monophosphate systems in the rat aorta. ANESTHESIOLOGY 40:162-167, 1974.
Su JY, Zhang CC: Intracellular mechanisms of halothane's effect on isolated aortic strips of the rabbit. ANESTHESIOLOGY 71:109-117, 1989.
Muldoon SM, Hart JL, Bowen KA, Freas W: Attenuation of endothelium-mediated vasodilation by halothane. ANESTHESIOLOGY 68:31-37, 1988.
Bollen BA, Tinker JH, Hermsmeyer K: Halothane relaxes previously constricted isolated porcine coronary artery segments more than isoflurane. ANESTHESIOLOGY 66:748-752, 1987.
Eskinder H, Rusch NJ, Supan FD, Kampine JP, Bosnjak ZJ: The effects of volatile anesthetics on 1.- and T-type calcium channel currents in canine cardiac Purkinje cells. ANESTHESIOLOGY 74:919-926, 1991.
Bosnjak ZJ, Supan FD, Rusch NJ: The effects of halothane enflurane and isoflurane on calcium current in isolated canine ventricular cells. ANESTHESIOLOGY 74:340-345, 1991.
Flynn NM, Buljubasic N, Bosnjak ZJ, Kampine JP: Cerebral vascular responses to anesthetics. Mechanisms of Anesthetic Action in Skeletal, Cardiac and Smooth Muscle Edited by Blanck TJJ, Wheeler DM. New York, Plenum, 1991, pp 237-246.
Langton PD, Nelson MT, Huang Y, Standen NB: Block of Calcium sup 2+ -activated Potassium sup + channels in mammalian arterial myocytes by tetraethylammonium ions. Am J Physiol 260:H927-H934, 1991.
Inoue R, Kitamura K, Kuriyama H: Two Calcium-dependent K-channels classified by the application of tetraethylammonium distribute to smooth muscle membranes of the rabbit portal vein. Pflugers Arch 405:173-179, 1985.
Stuenkel EL: Single potassium channels recorded from vascular smooth muscle cells. Am J Physiol 257:H760-H769, 1989.
Desilets M, Driska SP, Bumgarten C: Current fluctuations and oscillations in smooth muscle cells from hog carotid artery. Circ Res 65:708-722, 1989.
Nelson MT, Patlak JB, Worley JF, Standen NB: Calcium channels potassium channels, and voltage dependence of arterial smooth muscle tone. Am J Physiol 259:C3-C18, 1990.
Bonnet P, Rusch NJ, Harder DR: Characterization of an outward Potassium sup + current in freshly dispersed cerebral arterial muscle cells. Pflugers Arch 413:292-296, 1991.
Okabe K, Kitamura K, Kuriyama H: Features of 4-aminopyridine sensitive outward current observed in single smooth cells from rabbit pulmonary artery. Pflugers Arch 409:561-568, 1987.
Beech DJ, Bolton TB: Two components of potassium current activated by depolarization of single smooth muscle cells from the rabbit portal vein. J Physiol (Lond) 418:293-309, 1989.
Hirst GDS, Edwards FR: Sympathetic neuroeffector transmission in arteries and arteriole. Physiol Rev 69:546-604, 1989.
Standen NB, Quayle JM, Davies NW, Brayden JE, Huang Y, Nelson MT: Hyperpolarizing vasodilators activate ATP-sensitive Potassium sup + channels in arterial smooth muscle. Science 245:177-180, 1989.
Halsey MJ: Potency and physical properties of inhalational anaesthetics, General Anaesthesia. Part 1. Edited by Nunn JF, Utting JE, Brown BR Jr. London, Butterworths, 1989, pp 7-18.
Buljubasic N, Flynn NM, Marijic J, Rusch NJ, Kampine JP, Bosnjak ZJ: Effects of isoflurane on Potassium sup + and Calcium sup 2+ conductance in isolated smooth muscle cells of canine cerebral arteries. Anesth Analg 75:590-596, 1992.
Pancrazio JJ, Park WK, Lynch CL: Inhalational anesthetics actions on voltage-gated ion currents of bovine adrenal chromaffin cells. Mol Pharmacol 43:783-794, 1993.
Tas PWL, Kress HG, Koschel K: Volatile anesthetics inhibit the ion flux through Calcium sup 2+ -activated Potassium sup + channels of rat glioma C6 cells. Biochim Biophys Acta 983:264-268, 1989.
Antkowiak B, Kirschfeld: Enflurane is a potent inhibitor of high conductance Calcium sup 2+ -activated Potassium sup + channels of Chara australis. FEBS Lett 313:281-284, 1992.
Pancrazio JJ, Park WK, Lynch CL: Effects of enflurane on the voltage-gated membrane currents of bovine adrenal chromaffin cells. Neurosci Lett 146:147-151, 1992.
Vulliemoz Y, Verosky M and Trines L: Effect of halothane on myocardial cyclic AMP and cyclic GMP content of mice. J Pharmacol Exp Ther 236:181-186, 1985.
Eskinder H, Hillard CJ, Flynn N, Bosnjak ZJ, Kampine JP: Role of guanylate cyclase-cyclic GMP systems in halothane-induced vasodilation in canine cerebral arteries. ANESTHESIOLOGY 77:482-487, 1992.
Waldman SA, Murad F: Cyclic GMP synthesis and function, Pharmacol Rev 39:163-193, 1987.
Miller C: 1990: Annus mirabilis of potassium channel. Science 252:1092-1096, 1991.
Figure 1. Whole-cell outward Potassium sup + current in a single canine cerebral arterial muscle cell dialized with 1.8 mM CaCl2and 2.5 mM EGTA before (A) and after (B) addition of 3 mM tetraethylammonium (TEA). Cells were progressively depolarized from a holding potential of 60 mV to the corresponding potentials indicated on the pulse protocol. An outward current was elicited whose amplitude was markedly depressed by TEA. (C) Peak current-voltage relations obtained before and after exposure to 3 mM TEA in two canine cerebral arterial muscle cells.
Figure 1. Whole-cell outward Potassium sup + current in a single canine cerebral arterial muscle cell dialized with 1.8 mM CaCl2and 2.5 mM EGTA before (A) and after (B) addition of 3 mM tetraethylammonium (TEA). Cells were progressively depolarized from a holding potential of 60 mV to the corresponding potentials indicated on the pulse protocol. An outward current was elicited whose amplitude was markedly depressed by TEA. (C) Peak current-voltage relations obtained before and after exposure to 3 mM TEA in two canine cerebral arterial muscle cells.
Figure 1. Whole-cell outward Potassium sup + current in a single canine cerebral arterial muscle cell dialized with 1.8 mM CaCl2and 2.5 mM EGTA before (A) and after (B) addition of 3 mM tetraethylammonium (TEA). Cells were progressively depolarized from a holding potential of 60 mV to the corresponding potentials indicated on the pulse protocol. An outward current was elicited whose amplitude was markedly depressed by TEA. (C) Peak current-voltage relations obtained before and after exposure to 3 mM TEA in two canine cerebral arterial muscle cells.
×
Figure 2. Modulation of macroscopic Potassium sup + channel current by low (0.4 mM equivalent to 0.7%) and high (0.9 mM, equivalent to 1.5%) concentrations of halothane in canine cerebral arterial muscle cells dialized with 1.8 mM CaCl and 2.5 mM EGTA. (Top) The pulse protocol and actual recordings of macroscopic Potassium sup + current traces in control solution and after exposure to low or high doses of halothane. Voltage steps from a holding potential (HP) of 60 mV to the corresponding potentials elicited an outward current whose amplitude was depressed markedly and reversibly by halothane. (Bottom) The peak current-voltage relationship in control solution and after exposure to low and high doses of halothane, 0.4 (n = 4) and 0.9 mM (n = 5), respectively. Cells were depolarized progressively from a HP of 60 mV to the corresponding potentials indicated on the abscissa. Points with error bars represent the mean plus/minus SEM.
Figure 2. Modulation of macroscopic Potassium sup + channel current by low (0.4 mM equivalent to 0.7%) and high (0.9 mM, equivalent to 1.5%) concentrations of halothane in canine cerebral arterial muscle cells dialized with 1.8 mM CaCl and 2.5 mM EGTA. (Top) The pulse protocol and actual recordings of macroscopic Potassium sup + current traces in control solution and after exposure to low or high doses of halothane. Voltage steps from a holding potential (HP) of 60 mV to the corresponding potentials elicited an outward current whose amplitude was depressed markedly and reversibly by halothane. (Bottom) The peak current-voltage relationship in control solution and after exposure to low and high doses of halothane, 0.4 (n = 4) and 0.9 mM (n = 5), respectively. Cells were depolarized progressively from a HP of 60 mV to the corresponding potentials indicated on the abscissa. Points with error bars represent the mean plus/minus SEM.
Figure 2. Modulation of macroscopic Potassium sup + channel current by low (0.4 mM equivalent to 0.7%) and high (0.9 mM, equivalent to 1.5%) concentrations of halothane in canine cerebral arterial muscle cells dialized with 1.8 mM CaCl and 2.5 mM EGTA. (Top) The pulse protocol and actual recordings of macroscopic Potassium sup + current traces in control solution and after exposure to low or high doses of halothane. Voltage steps from a holding potential (HP) of 60 mV to the corresponding potentials elicited an outward current whose amplitude was depressed markedly and reversibly by halothane. (Bottom) The peak current-voltage relationship in control solution and after exposure to low and high doses of halothane, 0.4 (n = 4) and 0.9 mM (n = 5), respectively. Cells were depolarized progressively from a HP of 60 mV to the corresponding potentials indicated on the abscissa. Points with error bars represent the mean plus/minus SEM.
×
Figure 3. Whole-cell outward Potassium sup + current in a single canine cerebral arterial muscle cell dialized with zero Calcium2+ and 10 mM EGTA before (A) and after (B) addition of 1 mM 4-aminopyridine (4-AP). Cells were progressively depolarized from a holding potential of 60 mV to the corresponding potentials indicated on the pulse protocol. An outward current was elicited whose amplitude was markedly depressed by 4-AP. (C) Peak current-voltage relations obtained before and after exposure to 1 mM 4-AP in four canine cerebral arterial muscle cells. Points with error bars represent the mean plus/minus SEM.
Figure 3. Whole-cell outward Potassium sup + current in a single canine cerebral arterial muscle cell dialized with zero Calcium2+ and 10 mM EGTA before (A) and after (B) addition of 1 mM 4-aminopyridine (4-AP). Cells were progressively depolarized from a holding potential of 60 mV to the corresponding potentials indicated on the pulse protocol. An outward current was elicited whose amplitude was markedly depressed by 4-AP. (C) Peak current-voltage relations obtained before and after exposure to 1 mM 4-AP in four canine cerebral arterial muscle cells. Points with error bars represent the mean plus/minus SEM.
Figure 3. Whole-cell outward Potassium sup + current in a single canine cerebral arterial muscle cell dialized with zero Calcium2+ and 10 mM EGTA before (A) and after (B) addition of 1 mM 4-aminopyridine (4-AP). Cells were progressively depolarized from a holding potential of 60 mV to the corresponding potentials indicated on the pulse protocol. An outward current was elicited whose amplitude was markedly depressed by 4-AP. (C) Peak current-voltage relations obtained before and after exposure to 1 mM 4-AP in four canine cerebral arterial muscle cells. Points with error bars represent the mean plus/minus SEM.
×
Figure 4. Modulation of macroscopic Potassium sup + channel current by low (0.4 mM equivalent to 0.7%) and high (0.9 mM, equivalent to 1.5%) concentrations of halothane in canine cerebral arterial muscle cells dialized with zero Calcium2+ and 10 mM EGTA. (Top) The pulse protocol and actual recordings of macroscopic Potassium sup + current traces in control solution and after exposure to low or high doses of halothane. Voltage steps from a holding potential (HP) of 60 mV to the corresponding potentials elicited an outward current whose amplitude was depressed markedly and reversibly by halothane. (Bottom) The peak current-voltage relationship in control solution and after exposure to low and high doses of halothane, 0.4 (n = 6) and 0.9 mM (n = 6), respectively. Cells were depolarized progressively from a HP of 60 mV to the corresponding potentials indicated on the abscissa. Points with error bars represent the mean plus/minus SEM.
Figure 4. Modulation of macroscopic Potassium sup + channel current by low (0.4 mM equivalent to 0.7%) and high (0.9 mM, equivalent to 1.5%) concentrations of halothane in canine cerebral arterial muscle cells dialized with zero Calcium2+ and 10 mM EGTA. (Top) The pulse protocol and actual recordings of macroscopic Potassium sup + current traces in control solution and after exposure to low or high doses of halothane. Voltage steps from a holding potential (HP) of 60 mV to the corresponding potentials elicited an outward current whose amplitude was depressed markedly and reversibly by halothane. (Bottom) The peak current-voltage relationship in control solution and after exposure to low and high doses of halothane, 0.4 (n = 6) and 0.9 mM (n = 6), respectively. Cells were depolarized progressively from a HP of 60 mV to the corresponding potentials indicated on the abscissa. Points with error bars represent the mean plus/minus SEM.
Figure 4. Modulation of macroscopic Potassium sup + channel current by low (0.4 mM equivalent to 0.7%) and high (0.9 mM, equivalent to 1.5%) concentrations of halothane in canine cerebral arterial muscle cells dialized with zero Calcium2+ and 10 mM EGTA. (Top) The pulse protocol and actual recordings of macroscopic Potassium sup + current traces in control solution and after exposure to low or high doses of halothane. Voltage steps from a holding potential (HP) of 60 mV to the corresponding potentials elicited an outward current whose amplitude was depressed markedly and reversibly by halothane. (Bottom) The peak current-voltage relationship in control solution and after exposure to low and high doses of halothane, 0.4 (n = 6) and 0.9 mM (n = 6), respectively. Cells were depolarized progressively from a HP of 60 mV to the corresponding potentials indicated on the abscissa. Points with error bars represent the mean plus/minus SEM.
×
Figure 5. Effect of changes in pipette potential on single-channel current amplitude in cell-attached patches. (A) Representative tracings of single-channel activity taken at various pipette potentials (PP) in patches from two different cerebral arterial muscle cells, c - closed state of the channel. (B) The current-voltage (I-V) relationship for this channel obtained from six arterial membrane patches. The slope of the curve determined via linear regression analysis was 99 pS. Each point represents the mean plus/minus SEM.
Figure 5. Effect of changes in pipette potential on single-channel current amplitude in cell-attached patches. (A) Representative tracings of single-channel activity taken at various pipette potentials (PP) in patches from two different cerebral arterial muscle cells, c - closed state of the channel. (B) The current-voltage (I-V) relationship for this channel obtained from six arterial membrane patches. The slope of the curve determined via linear regression analysis was 99 pS. Each point represents the mean plus/minus SEM.
Figure 5. Effect of changes in pipette potential on single-channel current amplitude in cell-attached patches. (A) Representative tracings of single-channel activity taken at various pipette potentials (PP) in patches from two different cerebral arterial muscle cells, c - closed state of the channel. (B) The current-voltage (I-V) relationship for this channel obtained from six arterial membrane patches. The slope of the curve determined via linear regression analysis was 99 pS. Each point represents the mean plus/minus SEM.
×
Figure 6. Representative tracings showing concentration-dependent reduction by tetraethylammonium (TEA) of single-channel amplitude at pipette potential (PP) of -20 mV, c - closed state of the channel.
Figure 6. Representative tracings showing concentration-dependent reduction by tetraethylammonium (TEA) of single-channel amplitude at pipette potential (PP) of -20 mV, c - closed state of the channel.
Figure 6. Representative tracings showing concentration-dependent reduction by tetraethylammonium (TEA) of single-channel amplitude at pipette potential (PP) of -20 mV, c - closed state of the channel.
×
Figure 7. Representative traces showing the effect of Calcium2+ ionophore (A23187, 1 micro Meter) on single-channel activity at pipette potential (PP) of 0 mV, c - closed state of the channel.
Figure 7. Representative traces showing the effect of Calcium2+ ionophore (A23187, 1 micro Meter) on single-channel activity at pipette potential (PP) of 0 mV, c - closed state of the channel.
Figure 7. Representative traces showing the effect of Calcium2+ ionophore (A23187, 1 micro Meter) on single-channel activity at pipette potential (PP) of 0 mV, c - closed state of the channel.
×
Figure 8. Two single-channel current amplitudes recorded at pipette potential (PP) of 20 mV in two cell-attached patches from two different canine cerebral arterial muscle cells. (A) Channel activity showing small amplitude channel. (B) Channel activity showing large amplitude channel. c closed state of the channel.
Figure 8. Two single-channel current amplitudes recorded at pipette potential (PP) of 20 mV in two cell-attached patches from two different canine cerebral arterial muscle cells. (A) Channel activity showing small amplitude channel. (B) Channel activity showing large amplitude channel. c closed state of the channel.
Figure 8. Two single-channel current amplitudes recorded at pipette potential (PP) of 20 mV in two cell-attached patches from two different canine cerebral arterial muscle cells. (A) Channel activity showing small amplitude channel. (B) Channel activity showing large amplitude channel. c closed state of the channel.
×
Figure 9. Summary of statistical analysis of mean open time, open state probability (NPo), and event frequency (pipette potential -20 mV) single-channel Potassium sup + currents recorded from isolated canine middle cerebral arterial muscle cells before, during, and after halothane (0.9 mM, n - 9). All parameters were decreased significantly in the presence of halothane. This effect of halothane was reversed when the inflow perfusate was changed to the control solution. *Significant difference from control values at P < 0.05.
Figure 9. Summary of statistical analysis of mean open time, open state probability (NPo), and event frequency (pipette potential -20 mV) single-channel Potassium sup + currents recorded from isolated canine middle cerebral arterial muscle cells before, during, and after halothane (0.9 mM, n - 9). All parameters were decreased significantly in the presence of halothane. This effect of halothane was reversed when the inflow perfusate was changed to the control solution. *Significant difference from control values at P < 0.05.
Figure 9. Summary of statistical analysis of mean open time, open state probability (NPo), and event frequency (pipette potential -20 mV) single-channel Potassium sup + currents recorded from isolated canine middle cerebral arterial muscle cells before, during, and after halothane (0.9 mM, n - 9). All parameters were decreased significantly in the presence of halothane. This effect of halothane was reversed when the inflow perfusate was changed to the control solution. *Significant difference from control values at P < 0.05.
×
Figure 10. Representative traces of single-channel activity before, during, and after exposure to halothane (0.9 mM, equivalent to 1.5%) at pipette potential (PP) of 20 mV.c closed current state.
Figure 10. Representative traces of single-channel activity before, during, and after exposure to halothane (0.9 mM, equivalent to 1.5%) at pipette potential (PP) of 20 mV.c closed current state.
Figure 10. Representative traces of single-channel activity before, during, and after exposure to halothane (0.9 mM, equivalent to 1.5%) at pipette potential (PP) of 20 mV.c closed current state.
×
Figure 11. Representative traces of single-channel activity before, during, and after exposure to isoflurane (1.2 mM, equivalent to 2.7%) at pipette potential (PP) of 20 mV.c - closed channel state.
Figure 11. Representative traces of single-channel activity before, during, and after exposure to isoflurane (1.2 mM, equivalent to 2.7%) at pipette potential (PP) of 20 mV.c - closed channel state.
Figure 11. Representative traces of single-channel activity before, during, and after exposure to isoflurane (1.2 mM, equivalent to 2.7%) at pipette potential (PP) of 20 mV.c - closed channel state.
×
Table 1. Effect of TEA (1 and 3 mM) on 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)-Induced Vasodilation in Dog Cerebral Arterial Vessels
Image not available
Table 1. Effect of TEA (1 and 3 mM) on 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)-Induced Vasodilation in Dog Cerebral Arterial Vessels
×