Free
Meeting Abstracts  |   January 1999
Inhibitory Effects of Diazepam and Midazolam on Ca2+and K+Channels in Canine Tracheal Smooth Muscle Cells 
Author Notes
  • (Yamakage) Instructor in Anesthesiology.
  • (Matsuzaki, Tsujiguchi, Honma) Fellow in Anesthesiology.
  • (Namiki) Professor and Chair, Department of Anesthesiology.
Article Information
Meeting Abstracts   |   January 1999
Inhibitory Effects of Diazepam and Midazolam on Ca2+and K+Channels in Canine Tracheal Smooth Muscle Cells 
Anesthesiology 1 1999, Vol.90, 197-207. doi:
Anesthesiology 1 1999, Vol.90, 197-207. doi:
BENZODIAZEPINES, especially midazolam, have been used widely for sedation and to induce general anesthesia. In addition to their hypnotic action, these agents have a direct relaxing effect on airway smooth muscle [1,2] and vascular smooth muscle. [3,4] Because the intracellular concentration of free Ca2+([Ca2+]i) plays a central role in the regulation of airway smooth muscle tone, [5,6] a possible mechanism for the relaxation produced by benzodiazepines is a decrease in [Ca2+](i). Yoshimura et al. [7] used the Ca2+indicator fura-2 to show that relaxation of contracted porcine tracheal smooth muscle by midazolam at clinically relevant concentrations was associated with a decrease in [Ca2+]i. Sustained contraction of airway smooth muscle requires the continued entry of extracellular Ca2+, [8] and the blockade of voltage-dependent Ca2+channels (VDCCs) suppresses the sustained increase in [Ca2+]iin agonist-stimulated tracheal smooth muscle. [9] We hypothesized, therefore, that benzodiazepines reduce [Ca2+](i) by inhibiting VDCC.
Conversely, the open-state probability of VDCC depends on the plasma membrane potential, which is regulated by K+-selectivechannels. [10,11] One other potential mechanism for bronchodilation by benzodiazepines is enhanced K+conductance, leading to a decrease in VDCC opening and thus to muscle relaxation. [12] [Section]
In the current study, we used whole-cell patch-clamp techniques to identify the direct effects of the benzodiazepines diazepam and midazolam on Ca2+and K+channels in freshly dispersed canine tracheal smooth muscle cells. We also evaluated the antagonized effects of the benzodiazepine antagonists flumazenil and PK11195 (specific central-type and specific peripheral-type antagonists, respectively) on the Ca2+and K+channels.
Materials and Methods
Preparation of Dispersed Canine Tracheal Smooth Muscle Cells
The Sapporo Medical University Ethical Committee on Animal Research approved the study. Adult mongrel dogs weighing 9-12 kg were anesthetized with 10 mg/kg intramuscular ketamine and killed by exsanguination. The tracheas were excised quickly and placed in modified Krebs' solution equilibrated with 95% oxygen and carbon dioxide at 4 [degree sign]C (composed of 118 mM NaCl, 4.7 mM KCl, 21 mM NaHCO3, 1.2 mM MgSO (4), 1.2 mM KH2PO4, 10 mM glucose, and 2.5 mM CaCl2; pH [tilde operator] 7.4). Cells were dispersed according to previously described methods. [13,14] Briefly, tracheal smooth muscle was minced and incubated for 10 min in Ca2+-freemodified Tyrode's solution at room temperature (22-24 [degree sign]C). The modified Tyrode's solution contained 135 mM NaCl, 5.4 mM KCl, 1 mM MgCl2, 5 mM glucose, 5 mM N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid](HEPES), and 0.1%(wt/vol) bovine serum albumin, with the pH adjusted to 7.4 with 0.5 M tris-[hydroxymethy]aminomethane (Tris). The tissue was digested for 20 min at 37 [degree sign]C in Ca2+-freemodified Tyrode's solution that contained 0.08%(wt/vol) collagenase, 0.05% trypsin inhibitor, and 0.03% protease. Cells were dispersed by trituration, filtered through nylon mesh, and centrifuged. The pellet was resuspended in a modified Krafbruhe solution [15] and stored at 4 [degree sign]C for as long as 5 h before being used. The modified Kraftbruhe solution contained 85 mM KCl, 30 mM K2HPO4, 5 mM MgSO4, 5 mM Na2ATP, 5 mM pyruvic acid, 5 mM creatine, 20 mM taurine, 5 mM [small beta, Greek]-hydroxybutyrate, and 0.1%(wt/vol) fatty acid-free bovine serum albumin, with the pH adjusted to 7.25 with Tris.
Whole-cell Patch-clamp Recording
All experiments were performed at room temperature (22-24 [degree sign]C). Micropipettes were pulled from soda lime hematocrit tubing (GC-1.5; Narishige, Tokyo, Japan) using a two-stage puller (model PP-83, Narishige) and were heat polished. These had resistances of 3 to 5 m Omega when filled with solution. An aliquot (approximately 0.5 ml) of the cell suspension was placed in a perfusion chamber on the stage of an inverted microscope (IX-70; Olympus, Tokyo, Japan). At X600 magnification, a three-dimensional oil-driven micromanipulator (ONM-1; Narishige) was used to position the patch pipettes against the membrane of the tracheal smooth muscle cells. After obtaining a high-resistance seal (5-50 G Omega) with slight suction (5-20 cm water), the patch membrane was disrupted by strong negative pressure, which allowed the voltage of the entire cell membrane to be controlled [16] and permitted the pipette solution to diffuse into the cytoplasm. Membrane currents were monitored using a CEZ-2400 patch-clamp amplifier (Nihon Kohden, Tokyo, Japan), and the amplifier output was low-pass filtered at 2,000 Hz. Leak currents, estimated by appropriate scaling of currents during 20-mV hyperpolarizing pulses, were subtracted from each of these records. Membrane capacitance and series resistance were compensated for by using the internal circuitry of the patch-clamp amplifier. All data were digitized (10,000 samples per s), stored on a hard disk, and analyzed using a 8100/100AV Power Macintosh computer (Apple, Cupertino, CA) using the Pulse+PulseFit 8.02 and Igor Pro 2.04 analysis software programs (Heka, Wiesenstrasse, Lambrecht, Germany).
To measure inward Ca2+currents (ICa) through VDCCs, recording solutions were chosen to inhibit K+currents and enhance Ca (2+) currents. The pipette solution contained 130 mM CsCl, 4 mM MgCl2, 10 mM EGTA, 5 mM Na2ATP, and 10 mM HEPES, with the pH adjusted to 7.2 with Tris. The bath solution contained 130 mM tetraethylammonium chloride, 1 mM MgCl2, 10 mM CaCl2, 10 mM glucose, and 10 mM HEPES, with the pH adjusted to 7.4 with Tris. Whole-cell ICas were elicited at 5-s intervals by 150-ms depolarizing pulses (-50 to +40 mV in 10-mV increments) from a holding potential of -70 mV. Inactivation curves were determined using a double-pulse protocol that consisted of a 3-s prepulse to a potential of -70 to +20 mV, followed by a 150-ms depolarization to +20 mV. The peak change in the current during the test pulse was expressed as a fraction of that obtained with the -70-mV prepulse, and this quantity was fit to a Boltzmann expression [17,18] using least-squares analysis to estimate the potential of half-maximal inactivation (V1/2) and the slope factor (k).
To measure outward K+currents (IK), recording solutions were chosen to enhance the K+currents. The pipette solution contained 70 mM KCl, 60 mM K+-glutamate, 5 mM K2ATP, 1 mM MgCl2, 2.5 mM EGTA, 1.8 mM CaCl2, and 10 mM HEPES, with the pH adjusted to 7.2 with Tris; the computer-calculated [Ca2+]iwas [tilde operator] 10-6M. A variant of this solution contained 10 mM EGTA and no CaCl2, giving a [Ca2+]iof <or= to 10-9M. The bath solution contained 135 mM NaCl, mM KCl 5.2, 1.8 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, and 10 mM glucose, with the pH adjusted to 7.4 with Tris. Whole-cell IKs were elicited at 5-s intervals by 150-ms depolarizing pulses (-40 to +60 mV) from a holding potential of -70 mV.
Voltage-pulse protocols were performed in control solutions for more than 5 min to obtain a stable baseline. Cells were exposed to a single concentration of one of the benzodiazepines (diazepam, 10-8to 10-3M; or midazolam, 10-8to 10-3M) tested by changing the inflow perfusate of the chamber to one of a similar composition but with benzodiazepine. The perfusion chamber consisted of a glass coverslip bottom, with needles placed for rapid solution changes. The chamber volume was approximately 1 ml, and complete solution changes in the chamber could be obtained within 1 min using a peristaltic pump (CTP-3; Iuchi, Tokyo, Japan) attached to the input and output ports. After a 6-min exposure, the perfusate was switched again to the control solution. The G Omega seal was maintained for a period sufficient to evaluate the reversibility of the effects of benzodiazepine in 206 of 238 experiments (86%). In another experiment, the effects of the benzodiazepine antagonists flumazenil (10-5M, a specific central type [19]) and PK11195 (10-5M, a specific peripheral type [20,21]) on these channels were tested alone and with these benzodiazepine agonists.
To identify the characteristics of the ICaseen in this study, the effects of the L-type VDCC antagonist nifedipine (10-6M) and the agonist Bay K 8644 (10-6M) on ICawere evaluated. The effects of charybdotoxin (40 nM), a specific Ca2+-activatedK+(KCa) channel blocker, [22] and 4-aminopyridine (1 mM), a specific Ca2+-independentdelayed rectifier K+(KDR) channel blocker, [22] on IKwere also evaluated to identify the characteristics of the IKs seen in this study.
Materials
The following drugs and chemicals were used: trypsin inhibitor (from soybean), bovine serum albumin, Na2ATP, pyruvic acid, creatine, taurine, [small beta, Greek]-hydroxybutyrate, EGTA, TEACl, nifedipine, Bay K 8644, dimethyl sulfoxide, charybdotoxin, 4-aminopyridine (Sigma Chemical Co., St. Louis, MO), type-I collagenase (Gibco Laboratories, Grand Island, NY), protease (Calbiochem, La Jolla, CA), and PK11195 (Research Biochemicals, Natick, MA). Diazepam, midazolam, and flumazenil were donated by Yamanouchi Pharmaceutical Company (Tokyo, Japan). Nifedipine and Bay K 8644 were dissolved in ethanol, and diazepam was dissolved in dimethyl sulfoxide (0.01% final concentrations for both).
Statistical Analysis
Data are expressed as the mean +/− SD. The IC50s of the effects of the benzodiazepines on ICaand IKwere obtained using a Boltzmann expression. [17,18] Changes in peak whole-cell currents (ICaor IK) or in the inactivation parameters V1/2and k with exposure to each drug were compared at each applied potential using the paired, two-tailed t test. The percentage of control peak whole-cell currents (ICaor IK) and the values of V1/2and k after treatment were compared for the benzodiazepines using one-factor analysis of variance and a Kruskal-Wallis test. In all comparisons, P < 0.05 was considered significant.
Results
Electric Properties of Inward Ca2+Currents and the Effects of Benzodiazepines on the the Whole-cell Ca2+Currents
The ICaseen in enzymatically dispersed canine tracheal smooth muscle cells during step depolarizations from -70 mV peaked at approximately 10 ms and was inactivated with a time constant of approximately 50 to 90 ms (Figure 1A: control). During baseline conditions, threshold activation of ICaoccurred at approximately -20 mV, and maximum peak current amplitude was obtained at approximately +20 mV. In 138 cells, the maximum peak ICawas -318 +/− 26 pA (range, -201 to -612 pA). The inactivation parameters obtained in 28 cells during control conditions were V (1/2)=-20.4 +/− 2.9 mV and k = 7.2 +/− 1.3 mV. As previously reported in porcine tracheal smooth muscle cells, [13,14] the addition of 10-6M nifedipine, a blocker of slowly inactivating (L-type) Ca2+channels, virtually eliminated the ICaof canine tracheal smooth muscle cells by approximately 93%, and 10-6M Bay K 8644, an agonist of L-type Ca2+channels, enhanced the magnitude of ICa(by approximately 2.4 times) but did not alter the time course of the currents (n = 3 in each case, data not shown). Inward Ca2+currents with a similar time course were observed in the inactivation experiments.
Figure 1. The effects of midazolam on depolarization-induced whole-cell inward Ca2+current. (A) Typical recordings of the whole-cell inward Ca (2+) current induced by pulses as long as +20 mV in the absence and presence of 10-4M midazolam. The dashed line denotes no current. (B) Representative time course of peak ICaat +20 mV before and after exposure to 10-4M midazolam.
Figure 1. The effects of midazolam on depolarization-induced whole-cell inward Ca2+current. (A) Typical recordings of the whole-cell inward Ca (2+) current induced by pulses as long as +20 mV in the absence and presence of 10-4M midazolam. The dashed line denotes no current. (B) Representative time course of peak ICaat +20 mV before and after exposure to 10-4M midazolam.
Figure 1. The effects of midazolam on depolarization-induced whole-cell inward Ca2+current. (A) Typical recordings of the whole-cell inward Ca (2+) current induced by pulses as long as +20 mV in the absence and presence of 10-4M midazolam. The dashed line denotes no current. (B) Representative time course of peak ICaat +20 mV before and after exposure to 10-4M midazolam.
×
As shown in a representative trace for depolarization from -70 to +20 mV (Figure 1A), midazolam (10-4M) inhibited the magnitude of I (Ca) but did not obviously alter the time course of the current. Peak ICaobtained with repeated steps to +20 mV increased in a few minutes after obtaining the whole-cell configuration at time 0 to a stable plateau, decreased rapidly by approximately 50% during exposure to 10-4M midazolam and recovered completely with washout (Figure 1B). Similar results were obtained with diazepam. Figure 2shows the relation between peak ICaagainst applied potential before and after exposure to 10-6M diazepam and 10-5M midazolam. Each of these benzodiazepines significantly inhibited ICaat step potentials ranging from of -10 to +40 mV and decreased peak ICaat +20 mV by approximately 50%(n = 7). The actual percentage inhibitions of peak ICaachieved by these agents at these concentrations (50.3 +/− 8.4% and 45.8 +/− 8.8%, respectively) were not significantly different. There was no apparent shift in the voltage dependence of ICawith either of the benzodiazepines.
Figure 2. The relation between the peak whole-cell inward Ca2+current and applied potential before ([black circle], solid line) and after ([white circle], dashed line) exposure to the benzodiazepines 10-6M diazepam (A) and 10-5M midazolam (B). Symbols represent the mean +/− SD (n = 7, *P < 0.05).
Figure 2. The relation between the peak whole-cell inward Ca2+current and applied potential before ([black circle], solid line) and after ([white circle], dashed line) exposure to the benzodiazepines 10-6M diazepam (A) and 10-5M midazolam (B). Symbols represent the mean +/− SD (n = 7, *P < 0.05).
Figure 2. The relation between the peak whole-cell inward Ca2+current and applied potential before ([black circle], solid line) and after ([white circle], dashed line) exposure to the benzodiazepines 10-6M diazepam (A) and 10-5M midazolam (B). Symbols represent the mean +/− SD (n = 7, *P < 0.05).
×
We determined the dose dependence of the inhibition of peak ICaby each of these benzodiazepines. Figure 3shows the relation between the percentage of control peak ICaat +20 mV and the molar concentration of the agents in the bath solution. Each of the two benzodiazepines significantly inhibited peak ICain a dose-dependent manner. Midazolam (IC50= approximately 1.2 x 10-5M) required a 10-fold greater concentration to achieve the same inhibitory effect as that of diazepam (IC (50)= approximately 10-6M).
Figure 3. The relation between peak the whole-cell inward Ca2+current at +20 mV, expressed as a percentage of control, and the bath concentrations of the benzodiazepines diazepam ([black circle], solid line) and midazolam ([black square], dashed line). Symbols represent the mean +/− SD (n = 7). *P < 0.05, percentage comparison of control of peak the whole-cell inward Ca2+current without the agents. [dagger]P < 0.05, comparison of the values of midazolam at the same concentrations.
Figure 3. The relation between peak the whole-cell inward Ca2+current at +20 mV, expressed as a percentage of control, and the bath concentrations of the benzodiazepines diazepam ([black circle], solid line) and midazolam ([black square], dashed line). Symbols represent the mean +/− SD (n = 7). *P < 0.05, percentage comparison of control of peak the whole-cell inward Ca2+current without the agents. [dagger]P < 0.05, comparison of the values of midazolam at the same concentrations.
Figure 3. The relation between peak the whole-cell inward Ca2+current at +20 mV, expressed as a percentage of control, and the bath concentrations of the benzodiazepines diazepam ([black circle], solid line) and midazolam ([black square], dashed line). Symbols represent the mean +/− SD (n = 7). *P < 0.05, percentage comparison of control of peak the whole-cell inward Ca2+current without the agents. [dagger]P < 0.05, comparison of the values of midazolam at the same concentrations.
×
(Figure 4and Table 1) summarize the effects of the benzodiazepines diazepam and midazolam at equieffective inhibitory concentrations (10-6M and 10-5M, respectively) on the inactivation curves of ICa. Each of these agents shifted the inactivation curve to a more negative potential. The induced changes in V1/2brought about by these agents were statistically significant in each case, and there was no significant difference in V1/2between these agents. The slope factor k was not changed by exposure to either of the benzodiazepines.
Figure 4. The effects of the benzodiazepines diazepam (A) and midazolam (B) on voltage-dependent steady state inactivation of the whole-cell inward Ca (2+) current. The inactivation curves were generated under control conditions ([black circle], solid line) and then repeated in the presence of one of the benzodiazepines ([white circle], dashed line). Symbols represent the mean +/− SD (n = 7).
Figure 4. The effects of the benzodiazepines diazepam (A) and midazolam (B) on voltage-dependent steady state inactivation of the whole-cell inward Ca (2+) current. The inactivation curves were generated under control conditions ([black circle], solid line) and then repeated in the presence of one of the benzodiazepines ([white circle], dashed line). Symbols represent the mean +/− SD (n = 7).
Figure 4. The effects of the benzodiazepines diazepam (A) and midazolam (B) on voltage-dependent steady state inactivation of the whole-cell inward Ca (2+) current. The inactivation curves were generated under control conditions ([black circle], solid line) and then repeated in the presence of one of the benzodiazepines ([white circle], dashed line). Symbols represent the mean +/− SD (n = 7).
×
Table 1. Effects of the Benzodiazepines Diazepam and Midazolam on the Inactivation Parameters of Whole-cell Inward Ca2+Currents (ICa)
Image not available
Table 1. Effects of the Benzodiazepines Diazepam and Midazolam on the Inactivation Parameters of Whole-cell Inward Ca2+Currents (ICa)
×
The effects of the benzodiazepine antagonists flumazenil and PK11195 were also tested on the control ICaand on the inhibitory effect on ICaof the benzodiazepine agonists diazepam and midazolam. Flumazenil (10-5M) and PK11195 (10-5M) had no significant effect on the control ICa(n = 3 in each case, data not shown). Figure 5shows the time course of the peak ICaobtained in a representative cell with repeated steps to +20 mV during exposure to 10-5M flumazenil and 10-6M diazepam. Despite pretreatment with a high concentration of flumazenil, 10-6M diazepam still induced an approximate 50% inhibition. Similar results were obtained with 10-5M PK11195 and 10-6M diazepam, with 10-5M flumazenil and 10-5M midazolam, and with 10-5M PK11195 and 10-5M midazolam (n = 3 in each case, data not shown).
Figure 5. A representative time course of the peak whole-cell inward Ca2+current obtained with repeated steps to +10 mV during sequential additions of 10-5M flumazenil and 10-6M diazepam.
Figure 5. A representative time course of the peak whole-cell inward Ca2+current obtained with repeated steps to +10 mV during sequential additions of 10-5M flumazenil and 10-6M diazepam.
Figure 5. A representative time course of the peak whole-cell inward Ca2+current obtained with repeated steps to +10 mV during sequential additions of 10-5M flumazenil and 10-6M diazepam.
×
Electric Properties of Outward K+Currents and the Effects of Benzodiazepines on Them
(Figure 6A) shows a macroscopic outward K+current (IK) obtained from a freshly dispersed canine tracheal smooth muscle cell dialyzed with a pipette solution containing a [Ca2+]iof [tilde operator] 10-6M to enhance IKthrough KCachannels. The IKwas activated progressively by 150-ms depolarizing pulses from a holding potential of -70 mV to consecutively more positive membrane potentials. Stepwise depolarization from a holding potential of -70 mV to more than -30 mV elicited an outward IKwith a mean peak amplitude of 1,840 +/− 201 pA at +60 mV (n = 92). The addition of 40 nM charybdotoxin, a specific KCachannel blocker, significantly decreased peak IKwithout any change in the time course of the current (Figure 6B). Figure 6C summarizes the current-voltage (I-V) relation plotted as percentages of maximum IKbefore and after exposure to charybdotoxin. Application of charybdotoxin reduced the peak current at +60 mV by 65 +/− 14%(n = 4).
Figure 6. The whole-cell outward K+current in a canine tracheal smooth muscle cell dialyzed with 1.8 mM CaCl2and 2.5 mM EGTA before (A) and after (B) exposure to 40 nM charybdotoxin. The The whole-cell outward K (+) currents were generated by depolarizing pulses to -40, -20, 0, +20, +40, and +60 mV from a holding potential of -70 mV. (C) Relative peak current-voltage relations obtained before and after exposure to 40 nM charybdotoxin. Symbols represent the mean +/− SD (n = 4).
Figure 6. The whole-cell outward K+current in a canine tracheal smooth muscle cell dialyzed with 1.8 mM CaCl2and 2.5 mM EGTA before (A) and after (B) exposure to 40 nM charybdotoxin. The The whole-cell outward K (+) currents were generated by depolarizing pulses to -40, -20, 0, +20, +40, and +60 mV from a holding potential of -70 mV. (C) Relative peak current-voltage relations obtained before and after exposure to 40 nM charybdotoxin. Symbols represent the mean +/− SD (n = 4).
Figure 6. The whole-cell outward K+current in a canine tracheal smooth muscle cell dialyzed with 1.8 mM CaCl2and 2.5 mM EGTA before (A) and after (B) exposure to 40 nM charybdotoxin. The The whole-cell outward K (+) currents were generated by depolarizing pulses to -40, -20, 0, +20, +40, and +60 mV from a holding potential of -70 mV. (C) Relative peak current-voltage relations obtained before and after exposure to 40 nM charybdotoxin. Symbols represent the mean +/− SD (n = 4).
×
The effects of benzodiazepines on this charybdotoxin-sensitive I (K) was examined in 84 cells. Midazolam (10-4M) caused an approximately 15% reduction in IKwithout any apparent effect on the time course of the current (Figure 7A), an effect that was reversible when the drug was washed out (data not shown). Figure 7B shows the effects of 10 (-4) M midazolam on the current-voltage (I-V) relation for K+channel activation. This benzodiazepine significantly suppressed the IKamplitude over the entire voltage range studied without shifting the voltage dependency of the I-V relation. Figure 7C shows the relation between peak I (K) at +60 mV, expressed as a percentage control, and the bath concentrations of the benzodiazepines. Both the benzodiazepines diazepam and midazolam significantly and dose dependently inhibited IKbut required high concentrations of more than 10-5M and more than 10-4M, respectively, to show significant effects. Flumazenil (10-5M) and PK11195 (10-5M) had no effect on the control IKor on the inhibitory effect on the IKof these benzodiazepines (n = 3 in each case, data not shown).
Figure 7. The effects of midazolam on depolarization-induced whole-cell outward K+currents (IK) with a pipette solution including 1.8 mM CaCl2and 2.5 mM EGTA. (A) Typical recordings of IKinduced by pulses up to +60 mV in the absence and presence of 10-4M midazolam. The dashed line denotes no current. (B) Relative peak current-voltage relations obtained before and after exposure to 10-4M midazolam. (C) The relation between peak IKat +60 mV, expressed as a percentage of control, and the bath concentrations of the benzodiazepines diazepam ([black circle], solid line) and midazolam ([black square], dashed line). Symbols represent the mean +/− SD (n = 7). *P < 0.05, percentage comparison of control of peak IKwithout agents.
Figure 7. The effects of midazolam on depolarization-induced whole-cell outward K+currents (IK) with a pipette solution including 1.8 mM CaCl2and 2.5 mM EGTA. (A) Typical recordings of IKinduced by pulses up to +60 mV in the absence and presence of 10-4M midazolam. The dashed line denotes no current. (B) Relative peak current-voltage relations obtained before and after exposure to 10-4M midazolam. (C) The relation between peak IKat +60 mV, expressed as a percentage of control, and the bath concentrations of the benzodiazepines diazepam ([black circle], solid line) and midazolam ([black square], dashed line). Symbols represent the mean +/− SD (n = 7). *P < 0.05, percentage comparison of control of peak IKwithout agents.
Figure 7. The effects of midazolam on depolarization-induced whole-cell outward K+currents (IK) with a pipette solution including 1.8 mM CaCl2and 2.5 mM EGTA. (A) Typical recordings of IKinduced by pulses up to +60 mV in the absence and presence of 10-4M midazolam. The dashed line denotes no current. (B) Relative peak current-voltage relations obtained before and after exposure to 10-4M midazolam. (C) The relation between peak IKat +60 mV, expressed as a percentage of control, and the bath concentrations of the benzodiazepines diazepam ([black circle], solid line) and midazolam ([black square], dashed line). Symbols represent the mean +/− SD (n = 7). *P < 0.05, percentage comparison of control of peak IKwithout agents.
×
In a separate series of experiments, we used a pipette solution in which [Ca2+]iwas strongly buffered with 10 ma EGTA to minimize the outward IKthrough KCachannels, and we examined the effects of benzodiazepines on Ca2+-independentIK. Figure 8A shows a representative trace of IKunder these conditions. The IKs were activated progressively by 150-ms depolarizing pulses from a holding potential of -70 mV to consecutively more positive potentials. The mean peak amplitude in 92 cells was 518 +/− 110 pA at +60 mV. The application of 40 nM charybdotoxin had no effect on the IK(n = 3, data not shown). Then we examined the effect of 1 mM 4-aminopyridine on this Ca2+-independentI (K). As shown in Figure 8, B and C, 4-aminopyridine decreased the peak I (K) amplitude without changing the time course of the current at +60 mV by 72 +/− 14%.
Figure 8. Whole-cell outward K+current in a canine tracheal smooth muscle cell dialyzed with no CaCl2and 10 mM EGTA before (A) and after (B) exposure to 1 mM 4-aminopyridine. The whole-cell outward K+current values were generated by depolarizing pulses to -40, -20, 0, +20, +40, and +60 mV from a holding potential of -70 mV. (C) Relative peak current-voltage relations obtained before and after exposure to 1 mM 4-aminopyridine. Symbols represent the mean +/− SD (n = 4).
Figure 8. Whole-cell outward K+current in a canine tracheal smooth muscle cell dialyzed with no CaCl2and 10 mM EGTA before (A) and after (B) exposure to 1 mM 4-aminopyridine. The whole-cell outward K+current values were generated by depolarizing pulses to -40, -20, 0, +20, +40, and +60 mV from a holding potential of -70 mV. (C) Relative peak current-voltage relations obtained before and after exposure to 1 mM 4-aminopyridine. Symbols represent the mean +/− SD (n = 4).
Figure 8. Whole-cell outward K+current in a canine tracheal smooth muscle cell dialyzed with no CaCl2and 10 mM EGTA before (A) and after (B) exposure to 1 mM 4-aminopyridine. The whole-cell outward K+current values were generated by depolarizing pulses to -40, -20, 0, +20, +40, and +60 mV from a holding potential of -70 mV. (C) Relative peak current-voltage relations obtained before and after exposure to 1 mM 4-aminopyridine. Symbols represent the mean +/− SD (n = 4).
×
The effects of benzodiazepines on this 4-aminopyridine-sensitive I (K) were examined in 84 cells. Diazepam (10-4M) reversibly suppressed the IKwithout changing the time course of the current (Figure 9A). This agent significantly suppressed the IKamplitude for the entire voltage range studied without shifting the voltage dependency of the I-V relation (Figure 9B). Figure 9C shows the relation between the percentage control of peak IKat +60 mV and the bath concentrations of the benzodiazepines. Diazepam and midazolam significantly and dose dependently inhibited IKbut also needed high concentrations (>10-5M and >10 (-4) M, respectively) to depress IK. Flumazenil (10-5M) and PK11195 (10-5M) had no effect on the control IKor on the inhibitory effects on the IKof these benzodiazepines (n = 3 in each case, data not shown).
Figure 9. The effects of diazepam on depolarization-induced whole-cell outward K+current (IK) with a pipette solution including no CaCl (2) and 10 mM EGTA. (A) Typical recordings of IKinduced by pulses up to +60 mV in the absence and presence of 10-4M diazepam. The dashed line denotes no current. (B) Relative peak current-voltage relations obtained before and after exposure to 10-4M diazepam. (C) The relation between peak IKat +60 mV, expressed as a percentage of control, and the bath concentrations of the benzodiazepines diazepam ([black circle], solid line) and midazolam ([black square], dashed line). Symbols represent the mean +/− SD (n = 7). *P < 0.05, percentage comparison of control of peak IKwithout agents.
Figure 9. The effects of diazepam on depolarization-induced whole-cell outward K+current (IK) with a pipette solution including no CaCl (2) and 10 mM EGTA. (A) Typical recordings of IKinduced by pulses up to +60 mV in the absence and presence of 10-4M diazepam. The dashed line denotes no current. (B) Relative peak current-voltage relations obtained before and after exposure to 10-4M diazepam. (C) The relation between peak IKat +60 mV, expressed as a percentage of control, and the bath concentrations of the benzodiazepines diazepam ([black circle], solid line) and midazolam ([black square], dashed line). Symbols represent the mean +/− SD (n = 7). *P < 0.05, percentage comparison of control of peak IKwithout agents.
Figure 9. The effects of diazepam on depolarization-induced whole-cell outward K+current (IK) with a pipette solution including no CaCl (2) and 10 mM EGTA. (A) Typical recordings of IKinduced by pulses up to +60 mV in the absence and presence of 10-4M diazepam. The dashed line denotes no current. (B) Relative peak current-voltage relations obtained before and after exposure to 10-4M diazepam. (C) The relation between peak IKat +60 mV, expressed as a percentage of control, and the bath concentrations of the benzodiazepines diazepam ([black circle], solid line) and midazolam ([black square], dashed line). Symbols represent the mean +/− SD (n = 7). *P < 0.05, percentage comparison of control of peak IKwithout agents.
×
Discussion
Electric Properties of Inward Ca2+Currents and the Effects of Benzodiazepines on Them
As previously reported in porcine tracheal smooth muscle cells, [13,14] we measured depolarization-induced inward Ca2+currents (I (Ca)) in freshly dispersed canine tracheal smooth muscle cells under ionic conditions designed to inhibit K+and Na+currents and to enhance Ca2+currents. Based on their time and voltage dependencies, their sensitivity to a nifedipine blockade, and their enhancement by the Ca2+channel agonist Bay K 8644 (data not shown), these currents are presumed to reflect the activity of long-lasting (L-type) VDCCs. [13,14,23,24] 
Both of the benzodiazepines tested inhibited ICathrough VDCCs of canine tracheal smooth muscle cells without an apparent change in the kinetics of activation or inactivation (Figure 1A). The onset of inhibition was rapid and the effect was reversible (Figure 1B) and dose related (Figure 3). None of the benzodiazepines altered the voltage dependence of ICa(Figure 2), suggesting that the drug has no effect on membrane surface charge or on the voltage sensor of the channel. These data indicate a cellular effect of benzodiazepines that can account for the airway smooth muscle relaxant effects of these agents. [1,2,7] The concentrations of midazolam required to inhibit VDCCs shown in Figure 3are not similar to those required to directly relax preconstricted airway smooth muscle preparations. [1,2,25] This discrepancy may result from differences in species and concentrations of contractile agonists and from differences in the experimental techniques we used.
To evaluate the inhibitory actions of these benzodiazepines on VDCCs of tracheal smooth muscle cells further, we studied the effects of these agents on steady-state, voltage-dependent inactivation of ICa. During prolonged depolarization (prepulse), a fraction of the VDCCs enters an unavailable or “inactivated” state. The degree of steady state inactivation depends on the prepulse potential (Figure 4). [14,26] The mean potential of half-maximal inactivation (V1/2= approximately 20.4 mV) and the mean slope factor (k = 7.2 mV) that we obtained under control conditions are similar to values reported from porcine [14,26] and bovine [24] tracheal smooth muscle cells. Both of the benzodiazepines significantly shifted the inactivation curves to more negative potentials without changing the sigmoid shapes of the curves (Figure 4, Table 1). A qualitatively similar shift induced by a dihydropyridine-sensitive Ca2+antagonist, such as nifedipine, in canine colonic [27] and porcine tracheal [26] smooth muscle cells has been interpreted as evidence of a drug-induced stabilization of the inactivated state. [28] Because a phenylalkylamine Ca (2+) antagonist, such as verapamil, has no effect on the inactivation curve of ICa, [26] we speculate that benzodiazepines have a dihydropyridine-sensitive Ca2+antagonist-like inhibitory effect on VDCCs in canine tracheal smooth muscle cells, reflecting the stabilization of the VDCCs in their activated and inactivated states because of the high affinity of the agents for these states. [28] The reported resting membrane potential typically is approximately -50 to -60 mV for airway smooth muscle. [29] Based on the voltage-dependent blockade and a shift in inactivation of ICa, these agents would have a more inhibitory effect on ICaat a lower membrane potential.
Electric Properties of Outward K+Currents and the Effects of Benzodiazepines on Them
In this study, using 2.5 mM EGTA and 1.8 mM CaCl2([Ca2+]iof [tilde operator] 10-6M) in the pipette solution, we recorded whole-cell IKvalues in freshly dispersed canine tracheal smooth muscle cells. Because IKhad a large conductance, and peak IKat +60 mV was significantly suppressed by the KCachannel blocker [23] charybdotoxin by approximately 65%(Figure 6), this current may include a considerable amount of K+current through KCachannels. [30-32] Similar KCachannel blockers (charybdotoxin) or TEA-sensitive LKs have been observed in vascular smooth muscle cells. [33-35] In another study using a pipette solution containing no Ca2+and 10 mM EGTA to buffer the intracellular free Ca2+(Ca2+]iof <or= to 10 (-9) M), we also recorded a whole-cell outward IKthat was charybdotoxin insensitive but 4-aminopyridine sensitive (Figure 8). Under these conditions, IKdisplays the properties of IKthrough delayed rectifier K+(KDR) channels. [31-33] Similar outward KDRcurrents also have been observed in vascular smooth muscle. [35,36] 
Based on pharmacologic and electrophysiologic properties, these two types of K+channels have been identified in airway smooth muscle cell membranes. [32] The most common K+channel in airway smooth muscle is the KCachannel. [31,32] The open-state probability of this channel is low at the resting membrane potential but increases in proportion to membrane depolarization and elevation of [Ca2+]i. The resulting enhanced K+efflux will induce membrane repolarization or hyperpolarization, reduce the open-state probability of VDCCs, and in turn cause bronchodilation. In addition of this K+channel type, voltage- but not Ca2+-dependentKDRchannels also were identified in airway smooth muscle cell membranes. [32] The KDRchannels may play a central role in setting the level of the restine membrane potential. [32] 
In this study, the whole-cell outward IKs through KCaand KDRchannels were inhibited significantly by the two benzodiazepines tested (Figure 7and Figure 9), which might lead to membrane depolarization and muscle contraction. The results of this study indicate that diazepam and midazolam did not enhance the K+current, which would be expected to promote bronchodilation, but instead they reduced the activity of K+channels in canine tracheal smooth muscle cell membranes. This suggests that mechanisms other than K+channel opening are likely to mediate benzodiazepine-induced bronchodilation. Because these agents also needed high concentrations to inhibit IKs compared with those that exhibited significant inhibitory effects on ICas in canine tracheal smooth muscle cells (Figure 3, Figure 7, and Figure 9), the suppression of ICas through VDCCs may be one of the most important mechanisms by which benzodiazepines relax airway smooth muscle.
Interactions between Benzodiazepine Agonists and Antagonists on Whole-cell Inward Ca2+and Outward K+Currents
Airway smooth muscle tone also could be regulated by some neuropeptides. [37] [small gamma, Greek]-Aminobutyric acid (GABA) has an inhibitory effect on postganglionic cholinergic neurotransmission in ferret airways. [38] The benzodiazepine receptor is a positive modulatory subunit of the GABA receptor and enhances the chloride channel currents by increasing its opening frequency. [39] Therefore, in addition to directly inhibiting VDCCs, benzodiazepines might inhibit airway smooth muscle contraction by stimulating some benzodiazepine receptor, which leads to GABA receptor activation. In the current study, however, 10-5M flumazenil and 10-5M PK11195 (specific central [19] and specific peripheral [20,21] benzodiazepine antagonists, respectively) had no effect on the control ICaand IKor on the changes in ICaand IKinduced by the benzodiazepine agonists (Figure 5). Therefore, the diazepam and midazolam benzodiazepines probably relax the airway smooth muscle by binding cell membranes relating to VDCCs, rather than by activating benzodiazepine receptors. In support of our findings, studies have shown that flumazenil and PK11195 have no effect on benzodiazepine-induced relaxation of airway smooth muscle. [1,2,7] The concentration of flumazenil (10-5M) used in this study is greater than the estimated levels of plasma concentrations used clinically. [20,40,41] 
Concentration Dependence and Clinical Relevance
The benzodiazepines tested showed dose-dependent inhibition of I (Ca) and IK(Figure 3, Figure 7, and Figure 9). Diazepam is more potent than midazolam in terms of ICaand IK. Our data should be extrapolated to the clinical situation cautiously because of possible species differences, in vivo and in vitro differences, and the fact that our patch-clamp experiments were performed at low, nonphysiologic (ambient) temperature and using intracellular (pipette) and extracellular (organ bath) electrolytes. Nonetheless, the plasma concentrations of the benzodiazepines used clinically are approximately 3 x 10-7to 10-5M. [42-44] In the current study, the bath concentrations of the benzodiazepines diazepam and midazolam used to induce 50% inhibition of ICawere approximately 10 (-6) M and 10-5M, respectively, which seems relevant to clinical concentrations. We must note, however, that these agents are highly bound to plasma protein (>90% bound), [42] and the estimated plasma concentrations of free agents seem to be approximately 10-9to 10-6M. Therefore, the similarity between the clinical concentrations [42-44] and the concentrations needed to inhibit ICaby approximately 50% in this study simply may be a coincidence. Other mechanisms, such that benzodiazepines acts on the medulla to cause airway dilatation, [45] also should be considered.
In conclusion, the benzodiazepines diazepam and midazolam decreased the inward Ca2+current of canine tracheal smooth muscle cells, indicating the inhibition of VDCCs. This response could contribute to the ability of these agents to relax airway smooth muscle in vitro. A shift in the inactivation curve by these agents to more negative potentials can be interpreted as evidence of drug-induced stabilization of the inactivated state. Because the benzodiazepines reduced the K+channel activity at high concentrations, K+channel opening could not be responsible for the mechanisms of benzodiazepine-induced bronchodilation. The lack of antagonized effects of their antagonists flumazenil and PK11195 is related to the non-GABA-mediated electrophysiologic effects of benzodiazepines on airway smooth muscle contractility.
[Section] Ducros L, Plaisance P, Joseph T, Bard M, Salmeron S, Payen D, Lecarpentier Y: Determinants of ketamine-induce bronchodilation in guinea pig tracheal smooth muscle (abstract). Am J Respir Crit Care Med 1996; 153:A741
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Figure 1. The effects of midazolam on depolarization-induced whole-cell inward Ca2+current. (A) Typical recordings of the whole-cell inward Ca (2+) current induced by pulses as long as +20 mV in the absence and presence of 10-4M midazolam. The dashed line denotes no current. (B) Representative time course of peak ICaat +20 mV before and after exposure to 10-4M midazolam.
Figure 1. The effects of midazolam on depolarization-induced whole-cell inward Ca2+current. (A) Typical recordings of the whole-cell inward Ca (2+) current induced by pulses as long as +20 mV in the absence and presence of 10-4M midazolam. The dashed line denotes no current. (B) Representative time course of peak ICaat +20 mV before and after exposure to 10-4M midazolam.
Figure 1. The effects of midazolam on depolarization-induced whole-cell inward Ca2+current. (A) Typical recordings of the whole-cell inward Ca (2+) current induced by pulses as long as +20 mV in the absence and presence of 10-4M midazolam. The dashed line denotes no current. (B) Representative time course of peak ICaat +20 mV before and after exposure to 10-4M midazolam.
×
Figure 2. The relation between the peak whole-cell inward Ca2+current and applied potential before ([black circle], solid line) and after ([white circle], dashed line) exposure to the benzodiazepines 10-6M diazepam (A) and 10-5M midazolam (B). Symbols represent the mean +/− SD (n = 7, *P < 0.05).
Figure 2. The relation between the peak whole-cell inward Ca2+current and applied potential before ([black circle], solid line) and after ([white circle], dashed line) exposure to the benzodiazepines 10-6M diazepam (A) and 10-5M midazolam (B). Symbols represent the mean +/− SD (n = 7, *P < 0.05).
Figure 2. The relation between the peak whole-cell inward Ca2+current and applied potential before ([black circle], solid line) and after ([white circle], dashed line) exposure to the benzodiazepines 10-6M diazepam (A) and 10-5M midazolam (B). Symbols represent the mean +/− SD (n = 7, *P < 0.05).
×
Figure 3. The relation between peak the whole-cell inward Ca2+current at +20 mV, expressed as a percentage of control, and the bath concentrations of the benzodiazepines diazepam ([black circle], solid line) and midazolam ([black square], dashed line). Symbols represent the mean +/− SD (n = 7). *P < 0.05, percentage comparison of control of peak the whole-cell inward Ca2+current without the agents. [dagger]P < 0.05, comparison of the values of midazolam at the same concentrations.
Figure 3. The relation between peak the whole-cell inward Ca2+current at +20 mV, expressed as a percentage of control, and the bath concentrations of the benzodiazepines diazepam ([black circle], solid line) and midazolam ([black square], dashed line). Symbols represent the mean +/− SD (n = 7). *P < 0.05, percentage comparison of control of peak the whole-cell inward Ca2+current without the agents. [dagger]P < 0.05, comparison of the values of midazolam at the same concentrations.
Figure 3. The relation between peak the whole-cell inward Ca2+current at +20 mV, expressed as a percentage of control, and the bath concentrations of the benzodiazepines diazepam ([black circle], solid line) and midazolam ([black square], dashed line). Symbols represent the mean +/− SD (n = 7). *P < 0.05, percentage comparison of control of peak the whole-cell inward Ca2+current without the agents. [dagger]P < 0.05, comparison of the values of midazolam at the same concentrations.
×
Figure 4. The effects of the benzodiazepines diazepam (A) and midazolam (B) on voltage-dependent steady state inactivation of the whole-cell inward Ca (2+) current. The inactivation curves were generated under control conditions ([black circle], solid line) and then repeated in the presence of one of the benzodiazepines ([white circle], dashed line). Symbols represent the mean +/− SD (n = 7).
Figure 4. The effects of the benzodiazepines diazepam (A) and midazolam (B) on voltage-dependent steady state inactivation of the whole-cell inward Ca (2+) current. The inactivation curves were generated under control conditions ([black circle], solid line) and then repeated in the presence of one of the benzodiazepines ([white circle], dashed line). Symbols represent the mean +/− SD (n = 7).
Figure 4. The effects of the benzodiazepines diazepam (A) and midazolam (B) on voltage-dependent steady state inactivation of the whole-cell inward Ca (2+) current. The inactivation curves were generated under control conditions ([black circle], solid line) and then repeated in the presence of one of the benzodiazepines ([white circle], dashed line). Symbols represent the mean +/− SD (n = 7).
×
Figure 5. A representative time course of the peak whole-cell inward Ca2+current obtained with repeated steps to +10 mV during sequential additions of 10-5M flumazenil and 10-6M diazepam.
Figure 5. A representative time course of the peak whole-cell inward Ca2+current obtained with repeated steps to +10 mV during sequential additions of 10-5M flumazenil and 10-6M diazepam.
Figure 5. A representative time course of the peak whole-cell inward Ca2+current obtained with repeated steps to +10 mV during sequential additions of 10-5M flumazenil and 10-6M diazepam.
×
Figure 6. The whole-cell outward K+current in a canine tracheal smooth muscle cell dialyzed with 1.8 mM CaCl2and 2.5 mM EGTA before (A) and after (B) exposure to 40 nM charybdotoxin. The The whole-cell outward K (+) currents were generated by depolarizing pulses to -40, -20, 0, +20, +40, and +60 mV from a holding potential of -70 mV. (C) Relative peak current-voltage relations obtained before and after exposure to 40 nM charybdotoxin. Symbols represent the mean +/− SD (n = 4).
Figure 6. The whole-cell outward K+current in a canine tracheal smooth muscle cell dialyzed with 1.8 mM CaCl2and 2.5 mM EGTA before (A) and after (B) exposure to 40 nM charybdotoxin. The The whole-cell outward K (+) currents were generated by depolarizing pulses to -40, -20, 0, +20, +40, and +60 mV from a holding potential of -70 mV. (C) Relative peak current-voltage relations obtained before and after exposure to 40 nM charybdotoxin. Symbols represent the mean +/− SD (n = 4).
Figure 6. The whole-cell outward K+current in a canine tracheal smooth muscle cell dialyzed with 1.8 mM CaCl2and 2.5 mM EGTA before (A) and after (B) exposure to 40 nM charybdotoxin. The The whole-cell outward K (+) currents were generated by depolarizing pulses to -40, -20, 0, +20, +40, and +60 mV from a holding potential of -70 mV. (C) Relative peak current-voltage relations obtained before and after exposure to 40 nM charybdotoxin. Symbols represent the mean +/− SD (n = 4).
×
Figure 7. The effects of midazolam on depolarization-induced whole-cell outward K+currents (IK) with a pipette solution including 1.8 mM CaCl2and 2.5 mM EGTA. (A) Typical recordings of IKinduced by pulses up to +60 mV in the absence and presence of 10-4M midazolam. The dashed line denotes no current. (B) Relative peak current-voltage relations obtained before and after exposure to 10-4M midazolam. (C) The relation between peak IKat +60 mV, expressed as a percentage of control, and the bath concentrations of the benzodiazepines diazepam ([black circle], solid line) and midazolam ([black square], dashed line). Symbols represent the mean +/− SD (n = 7). *P < 0.05, percentage comparison of control of peak IKwithout agents.
Figure 7. The effects of midazolam on depolarization-induced whole-cell outward K+currents (IK) with a pipette solution including 1.8 mM CaCl2and 2.5 mM EGTA. (A) Typical recordings of IKinduced by pulses up to +60 mV in the absence and presence of 10-4M midazolam. The dashed line denotes no current. (B) Relative peak current-voltage relations obtained before and after exposure to 10-4M midazolam. (C) The relation between peak IKat +60 mV, expressed as a percentage of control, and the bath concentrations of the benzodiazepines diazepam ([black circle], solid line) and midazolam ([black square], dashed line). Symbols represent the mean +/− SD (n = 7). *P < 0.05, percentage comparison of control of peak IKwithout agents.
Figure 7. The effects of midazolam on depolarization-induced whole-cell outward K+currents (IK) with a pipette solution including 1.8 mM CaCl2and 2.5 mM EGTA. (A) Typical recordings of IKinduced by pulses up to +60 mV in the absence and presence of 10-4M midazolam. The dashed line denotes no current. (B) Relative peak current-voltage relations obtained before and after exposure to 10-4M midazolam. (C) The relation between peak IKat +60 mV, expressed as a percentage of control, and the bath concentrations of the benzodiazepines diazepam ([black circle], solid line) and midazolam ([black square], dashed line). Symbols represent the mean +/− SD (n = 7). *P < 0.05, percentage comparison of control of peak IKwithout agents.
×
Figure 8. Whole-cell outward K+current in a canine tracheal smooth muscle cell dialyzed with no CaCl2and 10 mM EGTA before (A) and after (B) exposure to 1 mM 4-aminopyridine. The whole-cell outward K+current values were generated by depolarizing pulses to -40, -20, 0, +20, +40, and +60 mV from a holding potential of -70 mV. (C) Relative peak current-voltage relations obtained before and after exposure to 1 mM 4-aminopyridine. Symbols represent the mean +/− SD (n = 4).
Figure 8. Whole-cell outward K+current in a canine tracheal smooth muscle cell dialyzed with no CaCl2and 10 mM EGTA before (A) and after (B) exposure to 1 mM 4-aminopyridine. The whole-cell outward K+current values were generated by depolarizing pulses to -40, -20, 0, +20, +40, and +60 mV from a holding potential of -70 mV. (C) Relative peak current-voltage relations obtained before and after exposure to 1 mM 4-aminopyridine. Symbols represent the mean +/− SD (n = 4).
Figure 8. Whole-cell outward K+current in a canine tracheal smooth muscle cell dialyzed with no CaCl2and 10 mM EGTA before (A) and after (B) exposure to 1 mM 4-aminopyridine. The whole-cell outward K+current values were generated by depolarizing pulses to -40, -20, 0, +20, +40, and +60 mV from a holding potential of -70 mV. (C) Relative peak current-voltage relations obtained before and after exposure to 1 mM 4-aminopyridine. Symbols represent the mean +/− SD (n = 4).
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Figure 9. The effects of diazepam on depolarization-induced whole-cell outward K+current (IK) with a pipette solution including no CaCl (2) and 10 mM EGTA. (A) Typical recordings of IKinduced by pulses up to +60 mV in the absence and presence of 10-4M diazepam. The dashed line denotes no current. (B) Relative peak current-voltage relations obtained before and after exposure to 10-4M diazepam. (C) The relation between peak IKat +60 mV, expressed as a percentage of control, and the bath concentrations of the benzodiazepines diazepam ([black circle], solid line) and midazolam ([black square], dashed line). Symbols represent the mean +/− SD (n = 7). *P < 0.05, percentage comparison of control of peak IKwithout agents.
Figure 9. The effects of diazepam on depolarization-induced whole-cell outward K+current (IK) with a pipette solution including no CaCl (2) and 10 mM EGTA. (A) Typical recordings of IKinduced by pulses up to +60 mV in the absence and presence of 10-4M diazepam. The dashed line denotes no current. (B) Relative peak current-voltage relations obtained before and after exposure to 10-4M diazepam. (C) The relation between peak IKat +60 mV, expressed as a percentage of control, and the bath concentrations of the benzodiazepines diazepam ([black circle], solid line) and midazolam ([black square], dashed line). Symbols represent the mean +/− SD (n = 7). *P < 0.05, percentage comparison of control of peak IKwithout agents.
Figure 9. The effects of diazepam on depolarization-induced whole-cell outward K+current (IK) with a pipette solution including no CaCl (2) and 10 mM EGTA. (A) Typical recordings of IKinduced by pulses up to +60 mV in the absence and presence of 10-4M diazepam. The dashed line denotes no current. (B) Relative peak current-voltage relations obtained before and after exposure to 10-4M diazepam. (C) The relation between peak IKat +60 mV, expressed as a percentage of control, and the bath concentrations of the benzodiazepines diazepam ([black circle], solid line) and midazolam ([black square], dashed line). Symbols represent the mean +/− SD (n = 7). *P < 0.05, percentage comparison of control of peak IKwithout agents.
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Table 1. Effects of the Benzodiazepines Diazepam and Midazolam on the Inactivation Parameters of Whole-cell Inward Ca2+Currents (ICa)
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Table 1. Effects of the Benzodiazepines Diazepam and Midazolam on the Inactivation Parameters of Whole-cell Inward Ca2+Currents (ICa)
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