Effects of Midazolam on Intracellular Calcium sup 2+ and Tension in Airway Smooth Muscles

Methods: Using front-surface fluorometry and fura-2-loaded porcine tracheal smooth muscle strips, both [Calcium²+]ⁱand isometric tension developments were simultaneously recorded.

Results: When the tracheal strips were exposed to a high external Potassium sup +-solution (40 mM) or 10 sup -7 M carbachol containing 1.25 mM Calcium²+, both [Calcium²+]ⁱand tension increased rapidly until they reached a plateau (the steady state). During steady-state contraction induced by Potassium sup +-depolarization or carbachol, the cumulative application of midazolam (10 sup -7 approximately 10 sub -4 M) caused decreases in both [Calcium²+]ⁱand tension, in a concentration-dependent manner. During 40 mM Potassium sup +-induced depolarization, the stepwise increases in the extracellular Calcium²+ concentration induced the stepwise increases in [Calcium sup 2+]ⁱand tension. Midazolam (3 x 10 sup -5 M) inhibited these increases in [Calcium²+]ⁱand tension, but had no effect on the [Calcium²+]ⁱ-tension relationship. In the presence of 3 x 10 sup -3 M NiCl²(a nonselective cation channel blocker), midazolam (3 x 10 sup -5 M) did not cause any additional reduction of [Calcium²+] sub i or tension during the contraction induced by carbachol (10 sup -7 M). In the absence of extracellular Calcium²+, midazolam (3 x 10 sup -5 M) had no effect on the transient increases in either [Calcium²+] sub i or the tension induced by carbachol (10 sup -7 M) or caffeine (20 mM). Pretreatment with both 10 sup -5 M flumazenil (a specific central antagonist of benzodiazepines) and 10 sup -5 M PK11195 (a specific peripheral antagonist of benzodiazepines) did not influence the effect of 10 sup -5 M midazolam on [Calcium²+]ⁱor tension during the contractions induced by carbachol.

Conclusions: Midazolam directly relaxes airway smooth muscles by decreasing [Calcium²+]ⁱ; this can be attributed to the inhibition of the influx of extracellular Calcium²+. Midazolam has no effect on the release of stored Calcium²+. In addition, midazolam has no effect on Calcium²+ sensitivity of the contractile apparatus. Finally, benzodiazepine antagonists, flumazenil and PK11195, have no effect on this mechanism of direct action of midazolam on airway smooth muscles.

MIDAZOLAM has been widely used as a sedative and an induction agent of general anesthesia. In addition to the hypnotic action, it has also been reported that midazolam causes vasodilation and relaxes airway smooth muscle. Midazolam's effect on the airway smooth muscle is favorable for patients who demonstrate airway hypersensitivity. However, there has been no report about the mechanism of the relaxant effects of midazolam on airway smooth muscle.

Although it is generally accepted that smooth muscle tone is primarily regulated by cytosolic Calcium²+ concentration ([Calcium sup 2+]ⁱ), the development and maintenance of tension does not simply depend on [Calcium²+]ⁱ. Recently, it has been shown that in alpha-toxin- or beta-escin-permeabilized smooth muscles, some agonists induced an enhanced sensitivity of myofilament to Calcium²+, through a G-protein-mediated pathway. In addition, it has also been shown that contractions can occur without any change in [Calcium²+] sub i in intact smooth muscle strips. Thus, to determine the mechanism underlying the changes in smooth muscle tone, it is important to determine the change in [Calcium²+]ⁱin association with the change in tension. In this study, we investigated the mechanism of midazolam-induced relaxation of airway smooth muscle by simultaneously measuring the tension and [Calcium²+]ⁱ, using front-surface fluorometry and the Calcium²+ indicator dye fura-2. The questions asked in this study were whether or not:(1) midazolam reduces [Calcium²+]ⁱ;(2) midazolam changes the sensitivity of the contractile apparatus to [Calcium²+]ⁱ; and (3) flumazenil (specific central benzodiazepine antagonist)or PK11195 (specific peripheral benzodiazepine antagonist)influence the effect of midazolam on airway smooth muscle.

Tracheas were dissected from adult pigs at a local slaughterhouse using a protocol approved by the Animal Research Committee of Research Institute of Angiocardiology, Faculty of Medicine, Kyushu University. The tracheas were placed in ice-cold physiologic salt solution (PSS) and brought to our laboratory. The lower end of the trachea, just above the first bronchus branching, three tracheal rings in length, was used for the experiments. The posterior portion of the trachea was excised longitudinally, and all cartilage was detached. The mucosa and adventitial tissue were removed under microscopic observation. The muscle sheets were cut transversely into rectangular strips approximately 3 mm long and 1 mm wide. All tissue preparations in the laboratory were performed in aerated PSS.

Tracheal strips were loaded with the Calcium²+ indicator dye, fura-2, in the form of acetoxymethyl ester (fura-2/AM). The strips were incubated in 1 ml aerated (95% O²: 5% CO²) Dulbecco-modified Eagle's medium containing 50 micro Meter Fura-2/AM and 5% fetal bovine serum for 3 hr at 37 degrees Celsius. After loading with fura-2, the strips were washed with normal PSS to remove dye in the extracellular space, and then were further incubated in normal PSS for at least 1 hr to facilitate the deesterification of intracellular fura-2/AM and to equilibrate the strips before taking the measurements. .

Each strip was mounted vertically in a 6-ml quartz organ bath, which was maintained at 37 degrees Celsius and bubbled with 95% O²and 5% CO². The lower end of the strip was fixed by a small clip and the upper end of the strip was attached by a small clip and thread to a force transducer (TB-612T, Nihon Koden, Japan) to record the isometric tension. During the 1-hr fura-2 equilibration period, the strips were stimulated with 40 mM Potassium sup + PSS at 5–10-min intervals, and muscle length was increased stepwise after each stimulation until the developed tension reached a maximum. When exposed to 40 mM Potassium sup + PSS, most strips produced stable tension within 15 min. The strips that showed an instability in tension, as induced by 40 mM Potassium sup + PSS, were excluded from the study. The responsiveness of each strip to 40 mM Potassium sup + PSS was then recorded before starting the experimental protocol, because almost the maximum, reproducible responses of tension to high Potassium sup + depolarization were obtained at this concentration of Potassium sup +. The developed tension was expressed as a percentage, assuming the values in normal (5.9 mM Potassium sup +) PSS and steady state of 40 mM Potassium sup + PSS to be 0% and 100%, respectively.

Changes in the fluorescence intensity of the fura-2-Calcium²+ complex were monitored using a front-surface fura-2 fluorometer (model CAM-OF Co.), specifically designed in collaboration with Japan Spectroscopic (Tokyo, Japan). The details of our front-surface fluorometry system have been described elsewhere. In brief, two wavelengths of excitation light (340 and 380 nm) were obtained spectroscopically from a Xenon light source. The strips were illuminated by guiding the two alternating (400-Hz) wavelengths of excitation light through quartz optic fibers. The surface fluorescence of the strip was collected by glass optic fibers and introduced through a 500-nm band-pass filter into a photomultiplier. Thus, we measured the fura-2 fluorescence intensity of 500-nm emission light, which was induced by alternating two wavelengths of excitation light (340 and 380 nm).

The ratio of the fluorescence intensities (fluorescence ratio) at 340 nm excitation to that at 380 nm excitation was monitored to estimate changes in [Calcium²+]ⁱand expressed as a percentage, assuming the values in normal PSS (5.9 mM Potassium sup +) and steady state of 40 mM Potassium sup + PSS to be 0% and 100%, respectively. The absolute values of [Calcium²+]ⁱfor 0% and 100% levels of the fluorescence ratio were determined separately using the following protocol and the equation of Grynkiewicz et al. : After recording 0% and 100% levels of the fluorescence ratio, the minimum and the maximum fluorescence ratios were determined by the addition of 25 micro Meter ionomycin to Calcium²+-free PSS containing 2 mM ethylene glycol-bis (beta-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA), followed by replacement with normal PSS (1.25 mM Calcium²+), respectively. The absolute values of [Calcium²+]ⁱin normal PSS (0%) and the steady state of 40 mM Potassium sup + PSS (100%) were calculated and they were 90 plus/minus 14 and 499 plus/minus 54 nM (n = 8), respectively. Thus, levels of [Calcium²+]ⁱfor each experiment were expressed as percent levels of the fluorescence ratio, and the absolute values of [Calcium²+]ⁱfor these percent levels were shown in the right ordinate of figures and parenthetically in the text as references.

To examine the effect of midazolam on [Calcium²+]ⁱand tension during contractions, midazolam (10 sup -7 approximately 10 sup -4 M) was cumulatively applied on the steady state of elevations of [Calcium sup 2+]ⁱand tension induced by 40 mM Potassium sup + PSS or 10 sup -7 M carbachol.

To examine the effect of antagonists against benzodiazepines on the effect of midazolam, strips were treated with flumazenil or PK11195. Flumazenil (10 sup -5 M) or PK11195 (10 sup -5 M) were applied for 5 min before and during the application of 10 sup -7 M carbachol. Midazolam (10 sup -5 M) was then applied at the steady state of the contraction induced by carbachol.

To examine the effect of midazolam on Calcium²+ sensitivity of the contractile apparatus, we determined the [Calcium²+]ⁱ-tension relationships in the contractions induced by the stepwise increases in the extracellular Calcium²+ concentration during 40 mM Potassium sup +-induced depolarization, in the absence or presence of 10 sup -7 M carbachol, as follows: After 10 min incubation in Calcium²+-free PSS containing 2 mM EGTA, and then 5 min incubation in Calcium²+ free PSS without EGTA, strips were immersed in Calcium sup 2+-free 40 mM Potassium sup + solution. Then, the extracellular Calcium²+ concentration was increased by the cumulative addition of CaCl². Midazolam (3 x 10 sup -5 M) was applied at the time of replacement with Calcium²+-free PSS without EGTA. To determine the effect of midazolam on the contractions in the presence of carbachol, 10 sup -7 M carbachol was applied after 5 min incubation in Calcium²+-free PSS containing 2 mM EGTA.

To examine the effect of midazolam on the dynamic changes in [Calcium²+]ⁱand tension during the contraction induced by carbachol, we observed the time courses of changes in [Calcium²+]ⁱand tension induced by 10 sup -7 M carbachol in strips being treated with midazolam (0, 3 x 10 sup -5 M) for 10 min before and during the application of carbachol.

To examine the effect of midazolam on Calcium²+ release from the intracellular store sites, two different experiments were performed, in which the Calcium²+ entry from the extracellular space was eliminated; the first is the inhibition of Calcium²+ influx and the second is the elimination of extracellular Calcium²+. First, the effect of midazolam on Calcium²+ release from the intracellular store during the inhibition of Calcium²+ influx was determined. To inhibit the influx of Calcium²+ from the extracellular space, 3 x 10 sup -3 M NiCl²(an inorganic Calcium²+ entry blocker ) was added 15 min before the application of 10 sup -7 M carbachol. Midazolam was applied 10 min before the application of carbachol. Second, the effect of midazolam on the Calcium²+ release by caffeine or carbachol was determined as follows; after 10 min incubation in Calcium²+-free PSS containing 2 mM EGTA, 20 mM caffeine or 10 sup -7 M carbachol was applied. In midazolam-treated strips, 3 x 10 sup -5 M midazolam was applied 5 min before and throughout the application of caffeine or carbachol.

The normal PSS consisted of the following composition (in mM): NaCl 123, KCl 4.7, NaHCO³15.5, KH²PO⁴1.2, CaCl²1.25, and D-glucose 11.5. High Potassium sup + PSS was identical to normal PSS, except for an equimolar substitution of KCl for NaCl. The Calcium²+-free version of PSS was produced by the exclusion of CaCl²from the composition of normal PSS. PSS was bubbled with 95% O²and 5% CO², with a resulting pH of 7.4 at 37 degrees Celsius. Fura-2/AM and EGTA were purchased from Dojindo (Kumamoto, Japan). The midazolam and flumazenil were donated by Yamanouchi Pharmaceutical Co. (Tokyo, Japan). The carbachol was obtained from Sigma Chemical (St. Louis, MO), and the caffeine was obtained from Katayama Chemical (Osaka, Japan), and the PK11195 was obtained from Research Biochemicals Inc. (Natick, MA).

The measured values were expressed as the mean plus/minus SE (n = number of observations). A repeated-measures one-way analysis of variance was used to determine the concentration-dependent effects. A repeated-measures two-way analysis of variance for was used to determine the statistical significance of the effect of midazolam pretreatment on the contractions induced by extracellularly applied Calcium²+ during high Potassium sup + depolarization. Analysis of covariance was used to determine the statistical significance of the shift of the [Calcium²+]ⁱ-tension relationship. For the rest of the measurements, an unpaired Student's t test was used. P values of less than 0.05 were considered to be significant. The IC⁵⁰values (the midazolam concentration that decreases [Calcium²+]ⁱand tension to 50% of the maximal response) were calculated, using the four-parameter logistic equation reported by De Lean et al. .

As shown in Figure 1(A), when the tracheal strip was depolarized by the exposure to 40 mM Potassium sup + PSS in the presence of 1.25 mM Calcium²+, [Calcium²+]ⁱand tension abruptly increased until reaching a peak, and then was sustained either at this level or at slightly decreased levels until reaching a plateau (or a steady state). When midazolam was cumulatively applied (10 sup -7 approximately 10 sup -4 M) during the steady state of the contraction, a concentration-dependent reduction in [Calcium²+]ⁱand tension occurred (F1-15(A and B)). The application of 10 sup -4 M midazolam reduced [Calcium²+]ⁱand tension to 18.1 plus/minus 5.2%(122 nM) and 6.5 plus/minus 2.1%, respectively (n = 6). The IC⁵⁰values for [Calcium²+]ⁱand tension were approximately 4.1 x 10 sup -5 M and 2.1 x 10 sup -5 M, respectively. When midazolam (10 sup -7 approximately 10 sup -4 M) was cumulatively applied during the resting state in normal PSS, no significant change in [Calcium²+]ⁱor tension was detected (data not shown). After washing out midazolam with normal PSS for 10 min, 40 mM Potassium sup + caused the same measure of response in [Calcium²+] sub i and tension, thereby indicating that the effect of midazolam (less or equal to 10 sup -4 M) examined in this study was reversible.

(Figure 2(A)) is a representative recording showing the effect of a cumulative application of midazolam (10 sup -7 approximately 6 x 10 sup -5 M) on [Calcium²+]ⁱand tension during the contraction induced by 10 sup -7 M carbachol. After recording the 100% response levels by the depolarization with 40 mM Potassium sup + PSS, carbachol was applied. Carbachol induced a rapid rise (the first component) in [Calcium²+] sub i and tension followed by a gradual decrease to a steady-state level (the second component) within 10 min. These steady levels of [Calcium²+]ⁱ[58.5 plus/minus 9%(259.1 nM)] and tension (118 plus/minus 12%)(n = 5) were then maintained during the 1-hr observation period. The cumulative application of midazolam, at the steady level of contraction induced by carbachol, caused a concentration-dependent reduction in [Calcium²+]ⁱand tension. The application of 6 x 10 sup -5 M midazolam reduced [Calcium²+]ⁱand tension to 2.31 plus/minus 0.9%(95.1 nM) and 4.1 plus/minus 1.1%, respectively (n = 6). The IC⁵⁰values for [Calcium²+]ⁱand tension were approximately 6.5 x 10 sup -6 M and 6.3 x 10 sup -6 M, respectively.

(Figure 3(A)) is a representative recording of the effect of application of 10 sup -5 M midazolam on elevated [Calcium²+]ⁱand tension induced by 10 sup -7 M carbachol. [Calcium²+]ⁱand tension were rapidly reduced to reach steady-state levels by midazolam. Neither the treatment with 10 sup -5 M flumazenil (F3-15(B)) nor the treatment with 10 sup -5 M PK11195 (F3-15(C)) for 5 min before and during the application of carbachol influenced carbachol-induced elevation of [Calcium²+]ⁱand tension, nor did it effect midazolam-induced reduction of elevated [Calcium²+]ⁱand tension (F3-15(D and E)).

(Figure 4(A)) shows a representative recording of changes in [Calcium sup 2+]ⁱand tension induced by the cumulative application of CaCl sub 2 during depolarization with 40 mM Potassium sup +. In response to the stepwise increment of extracellular Calcium²+ concentration (0.0125–2.5 mM), [Calcium²+]ⁱand tension increased in a concentration-dependent manner. When the extracellular Calcium²+ was 2.5 mM, [Calcium²+}i and tension were 105.6 plus/minus 7.2%(548 nM) and 85.3 plus/minus 10.5%, respectively (n = 10). Treatment with 3 x 10 sup -5 M midazolam for 8 min before and during the cumulative application of extracellular Calcium²+ significantly inhibited increases in [Calcium²+]ⁱand tension (P < 0.01 for both, by two-way analysis of variance)(F4-15(B)). In the midazolam-treated strips, when the extracellular Calcium²+ was 5 mM, [Calcium²+]ⁱand tension were 61.8 plus/minus 8.6%(273.6 nM) and 20.8 plus/minus 8.2% respectively (n = 6).

(Figure 5(A)) shows a representative recording of changes in [Calcium sup 2+]ⁱand tension induced by the cumulative application of CaCl sub 2 during depolarization with 40 mM Potassium sup + in the presence of 10 sup -7 M carbachol. In response to the stepwise increment of extracellular Calcium²+ concentration (0.0125–1.25 mM), [Calcium²+]ⁱand tension increased in a concentration-dependent fashion. When the extracellular Calcium²+ was 1.25 mM, [Calcium²+]ⁱand tension were 72.4 plus/minus 8.2%(322.8 nM) and 206.3 plus/minus 14.2%, respectively (n = 10). Treatment with 3 x 10 sup -5 M midazolam for 8 min before and during the cumulative application of extracellular Calcium²+ significantly inhibited increases in [Calcium²+]ⁱand tension (P < 0.01 for both, by two-way analysis of variance;F5-15(B)). In the midazolam-treated strips, when the extracellular Calcium²+ was 2.5 mM, [Calcium²+]ⁱand tension were 55.5 plus/minus 7.6%(247.8 nM) and 129.3 plus/minus 14.3%, respectively (n = 6).

(Figure 6) represents the [Calcium²+]ⁱ-tension relationships during the contractions induced by cumulative applications of the extracellular Calcium²+ during high Potassium sup + depolarization, in the presence or absence of carbachol, with or without midazolam treatment, were evaluated from data in F4-15and F5-15. The [Calcium²+]ⁱ-tension relationship of the contractions induced by the increases in the extracellular Calcium²+ concentration during high Potassium sup + depolarization in the presence of carbachol significantly shifted upward and left from that in the absence of carbachol. Treatment with 3 x 10 sup -5 M midazolam had no significant effect on the [Calcium²+]ⁱ-tension relationship both in the absence and presence of carbachol.

As shown in Figure 7, increases in both [Calcium²+]ⁱand tension induced by 10 sup -7 M carbachol were inhibited by pretreatment with midazolam (3 x 10 sup -5 M) for 10 min. Midazolam inhibited both [Calcium²+]ⁱand tension in a parallel manner. NiCl²(3 x 10 sup -3 M) only partially inhibited the first component, but almost fully inhibited the second component of the increases in [Calcium²+] sub i and tension during the contraction induced by 10 sup -7 M carbachol. At 3 x 10 sup -5 M midazolam alone, the extent of the increases in [Calcium²+]ⁱand tension of the first component were similar to those induced by 10 sup -7 M carbachol in strips pretreated with 3 x 10 sup -3 M NiCl²for 5 min, or to that pretreated with 3 x 10 sup -3 M NiCl²and 3 x 10 sup -5 M midazolam. The time courses of the changes in both [Calcium²+]ⁱand tension during the contraction induced by 10 sup -7 M carbachol in strips treated with 3 x 10 sup -3 M NiCl²were exactly the same as those in strips treated with 3 x 10 sup -3 M NiCl²and 3 x 10 sup -5 M midazolam, which indicated that midazolam could only inhibit Calcium²+ influx in a manner similar to that of nickel.

(Figure 8(A)) shows the representative recordings of [Calcium²+] sub i and tension of the contraction induced by the application of 20 mM caffeine in Calcium²+-free solution containing 2 mM EGTA. The application of 20 mM caffeine in the absence of extracellular Calcium²+ caused a transient increase in [Calcium²+]ⁱand tension. Treatment with 3 x 10 sup -5 M midazolam from 5 min before the application of caffeine had no effect on the increases in [Calcium²+]ⁱand tension induced by caffeine (F8-15(B and C)).

(Figure 9(A)) shows representative recordings of [Calcium²+]ⁱand tension of the contraction induced by the application of 10 sup -7 M carbachol in Calcium²+-free solution containing 2 mM EGTA. The application of 10 sup -7 M carbachol in the absence of extracellular Calcium²+ caused a transient increase in [Calcium²+]ⁱand tension. Treatment with 3 x 10 sup -5 M midazolam from 5 min before the application of carbachol had no effect on the increases in [Calcium²+]ⁱand tension induced by carbachol (F9-15(B and C)).

Several investigators have reported that midazolam directly relaxed smooth muscle during contractions induced by high Potassium sup + depolarization or agonists, including acetylcholine, bethanechol, histamine, and serotonin. However, the underlying intracellular mechanism of the direct relaxation of smooth muscle, especially that of airway smooth muscle, induced by midazolam has not yet been fully determined. On the basis of the current understanding of excitation-contraction coupling in smooth muscle cells, the following two mechanisms may play a major role: those dependent on changes in the surface membrane potential (electromechanical coupling) and those independent of the surface membrane potential (pharmacomechanical coupling). The independence (or dependence) of these two coupling mechanisms is not defined yet. Through these mechanisms, the increased cytosolic Calcium²+ binds to calmodulin to activate myosin light chain kinase, which catalyzes the phosphorylation of the myosin light chain, activating actomyosin adenosine triphosphatase, which induces contractions. Thus, [Calcium²+]ⁱprimarily regulates smooth muscle contractions through electromechanical and pharmacomechanical couplings. However, in smooth muscle cells, contractile force does not simply depend on [Calcium²+]ⁱ, because there are modulations of the sensitivity of myofilament to [Calcium²+]ⁱby several intracellular signal transduction systems; and this mechanism also may be included in pharmacomechanical coupling. To clarify the mechanism of the midazolam-induced relaxation of airway smooth muscle, we determined the effect of midazolam on electromechanical and pharmacomechanical coupling, namely, on Calcium²+ influx through the voltage-operated or receptor-operated Calcium²+ channels, on Calcium sup 2+ release from the intracellular store, and on the Calcium²+ sensitivity of the contractile apparatus.

In the current study at the steady state of both high Potassium sup + depolarization-induced and carbachol-induced contractions, cumulative applications of midazolam (10 sup -7 approximately 10 sup -4 M) caused a concentration-dependent decrease in [Calcium²+]ⁱand tension. Treatment with 3 x 10 sup -5 M midazolam inhibited the extracellularly applied Calcium²+-induced increases in [Calcium²+]ⁱand tension during high Potassium sup + depolarization, in the absence or presence of carbachol. However, at the resting state, the cumulative application of midazolam (10 sup -7 approximately 10 sup -4 M) led to no significant change in either [Calcium²+]ⁱor tension. These results suggest that midazolam inhibits the influx of extracellular Calcium²+ induced both by high Potassium sup + depolarization and carbachol and, thus, decreases [Calcium²+]ⁱto cause relaxation during contractions.

Airway contraction is controlled by some neuropeptides. Gamma-amino-butyric acid has an inhibitory effect on postganglionic cholinergic neurotransmission in ferret airways. The benzodiazepine receptor is a positive modulatory subunit of gamma-amino-butyric acid receptor. A ligand bound to the benzodiazepine receptor enhances the effect of gamma-amino-butyric acid on the chloride channel by increasing its opening frequency. Therefore, it is possible that, in addition to the direct inhibition of a Calcium²+ influx possibly through Calcium²+ channels, midazolam relaxes airway smooth muscle by means of stimulation of some benzodiazepine receptor and modulation of the gamma-amino-butyric acid receptor. In the current study, however, 10 sup -5 M flumazenil and PK11195 (a specific central and a specific peripheral antagonist against benzodiazepine, respectively), had no influence on the changes in [Calcium²+]ⁱor tension during 10 sup -5 M midazolam-induced relaxation. Midazolam probably relaxes the airway smooth muscle by binding sarcolemmal membranes relating to Calcium sup 2+ channels, but not by activating benzodiazepine receptors. In agreement with our findings, flumazenil and PK11195 have been reported to have no effect on midazolam-induced relaxation of airway smooth muscle. The concentration of flumazenil (10 sup -5 M) used in the current study is higher than the estimated levels of plasma concentrations in clinical use. .

In the presence of carbachol, the [Calcium²+]ⁱ-tension relation-curve of the contractions induced by the increases in extracellular Calcium²+ concentration during high Potassium sup +-depolarization, shifted markedly upward and left from that obtained in the absence of carbachol. In other words, at a given [Calcium²+]ⁱlevel, the tension development in the presence of carbachol was much greater than that in the absence of carbachol, indicating that the Calcium sup 2+ sensitivity of the contractile apparatus is increased by carbachol. Similar findings, that muscarinic agonists increase myofilament Calcium sup 2+ sensitivity in airway smooth muscle, have been reported by others. In addition, the current study showed that, not only the Calcium²+ sensitivity during the contraction induced by high Potassium sup +-depolarization but also an increased Calcium²+ sensitivity of the contractile apparatus induced by carbachol is not affected by the midazolam treatment. These findings are essentially similar to those observed in the case of diltiazem and indicated that the relaxation of the carbachol-induced contractions by midazolam is directly caused by the decrease in [Calcium²+]ⁱ, without any direct effect on the Calcium²+ sensitivity of the contractile apparatus.

In the current study, the application of carbachol (10 sup -7 M) on porcine tracheal strips increased both [Calcium²+]ⁱand tension, which consisted of two components: the first, a rapid rising component and the second, a sustained component (F7-15). Since the application of carbachol, both in the absence of extracellular Calcium²+ and the presence of both Calcium²+ and Nickel²+, caused only rapid and transient increases in [Calcium²+]ⁱand tension, which was smaller than that seen with the presence of extracellular Calcium²+(F7-15and F9-15), it is thus conceivable that the second component is produced mainly by the influx of extracellular Calcium²+, whereas the first component is produced by both the Calcium²+ influx and Calcium²+ release from the intracellular store. Because pretreatment with midazolam (3 x 10 sup -5 M) attenuated the carbachol-induced elevations of [Calcium²+]ⁱand tension in both the first and second components, we carried out additional experiments to determine the effect of midazolam on the release of Calcium²+ from the intracellular store. The following major results were obtained: First, when carbachol was applied in the presence of NiCl²(3 x 10 sup -3 M) to inhibit the Calcium²+ influx from the extracellular spaces, midazolam (3 x 10 sup -5 M) did not affect the elevation of [Calcium²+]ⁱand tension of the first component at all. Second, the effect of midazolam on the Calcium²+ release by two distinct stimulants, caffeine and carbachol, in the absence of extracellular Calcium²+ was examined. It is well established that caffeine causes Calcium²+ release by facilitating the Calcium²+-induced Calcium²+ release mechanism, whereas carbachol causes Calcium²+ release by pharmacomechanical coupling mediated by inositol 1,4,5,-trisphosphate. In the absence of extracellular Calcium²+, 3 x 10 sup -5 M midazolam was not found to have any inhibiting effect on the increases in [Calcium²+]ⁱand tension induced by either caffeine or carbachol. We found that only an extremely high concentration of midazolam (3 x 10 sup -4 M) inhibited the increases in [Calcium²+]ⁱand tension due to the release of Calcium²+ from the intracellular store induced by either caffeine or carbachol. However, the effects of midazolam, at a concentration higher than this level was not reversible (data not shown). These observations indicate that the clinical concentrations of midazolam do not affect Calcium²+ release from the intracellular stores.

The blood concentration of benzodiazepines used clinically is 100 approximately 200 micro gram/ml, i.e., approximately 10 sup -7 approximately 10 sup -6 M. In the current study, to observe the definite relaxation with midazolam during the reproducible and steady-state levels (118 plus/minus 12%) of contraction of tracheal smooth muscle, carbachol at the concentration of 10 sup -7 M was used an agonist. The IC⁵⁰value of midazolam for this high level of developed tension induced by 10 sup -7 M carbachol was 6.3 x 10 sup -6 M, which seems relevant to the clinical concentration. However, it has to be noted that because midazolam is highly bound to plasma protein and the tension development over 100% levels (like a contraction) hardly occur in the clinical setting, the similarity between the clinical concentration and the IC⁵⁰values obtained in the current study may be simply a coincidence.

Using front-surface fluorometry of fura-2 and porcine tracheal smooth muscle specimens, we were able to determine the mechanisms underlying midazolam-induced relaxation. It was concluded that midazolam (less or equal to 10 sup -4 M) directly inhibited extracellular Calcium sup 2+-dependent increases in [Calcium²+]ⁱ, possibly by means of a Calcium²+ influx through Calcium²+ channels, and thus, caused proportional decreases in tension, while having no effect on the Calcium²+ sensitivity of the contractile elements.