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Pain Medicine  |   February 2002
Effects of Primary Alcohols on Airway Smooth Muscle
Author Affiliations & Notes
  • Chie Sakihara, M.D.
    *
  • Keith A. Jones, M.D.
  • Robert R. Lorenz, B.S.
  • William J. Perkins, M.D.
    §
  • David O. Warner, M.D.
  • * Research Fellow, Department of Anesthesiology, † Associate Professor of Anesthesiology, ‡ Research Associate, § Assistant Professor of Anesthesiology, ∥ Professor of Anesthesiology, Departments of Anesthesiology and Physiology and Biophysics, Mayo Clinic and Mayo Foundation.
  • Received from the Departments of Anesthesiology and Physiology and Biophysics, Mayo Clinic and Mayo Foundation, Rochester, Minnesota.
Article Information
Pain Medicine
Pain Medicine   |   February 2002
Effects of Primary Alcohols on Airway Smooth Muscle
Anesthesiology 2 2002, Vol.96, 428-437. doi:
Anesthesiology 2 2002, Vol.96, 428-437. doi:
VOLATILE anesthetics are potent bronchodilators. They relax airway smooth muscle (ASM) by both depressing the function of nerves innervating ASM and by directly affecting the ASM itself. 1–3 This latter effect is caused by decreases in both concentration of intracellular calcium ([Ca2+]i) 4–6 and the force produced for a given [Ca2+]i(i.e.  , the Ca2+sensitivity) during membrane receptor stimulation. 4–7 These actions are mediated by a variety of mechanisms, including effects on ion channels, calcium storage and release within the cell, and second messengers within the cells, such as heterotrimeric guanine nucleotide-binding proteins (G-proteins). 8,9 Although these general categories of mechanism are well-established, the precise nature of the interaction between these targets and the anesthetics that produce these effects is not known. It appears that an action at all these sites is not a general property of anesthetics. For example, although other anesthetic agents such as propofol, midazolam, and ketamine relax ASM and share the ability of volatile anesthetics to decrease [Ca2+]i, they do not affect Ca2+sensitivity. 10 
One venerable approach to exploring anesthetic mechanisms of action has been to utilize homologous families of anesthetics to probe structure–activity relations in model systems. The primary alcohols have been extensively studied for this purpose, both in living animals and in model systems such as firefly luciferase. 11,12 Their anesthetic potency increases with chain length, with a cutoff in potency beyond dodecanol. Their effects on minimum alveolar concentration (MAC) are approximately additive with the volatile anesthetics, consistent with a similar mechanism of action. 11,12 In experimental systems, they have the advantage of being relatively soluble and stable in aqueous solution. In such experimental systems, the primary alcohol ethanol appears to have effects on putative targets of volatile anesthetic action such as ion channels and responses to membrane stimulation mediated by G-proteins, 13–16 although its potency in producing these effects is low.
We explored whether primary alcohols could be used as tools to explore the mechanism of anesthetic actions in ASM. We tested the hypothesis that, like volatile anesthetics, the primary alcohols relax ASM by decreasing [Ca2+]iand inhibiting agonist-induced increases in Ca2+sensitivity.
Materials and Methods
Tissue Preparation
After approval by the Mayo Institutional Animal Care and Use Committee, mongrel dogs of either sex were anesthetized with an intravenous injection of pentobarbital sodium (30 mg/kg) and exsanguinated. The extrathoracic trachea was excised and immersed in chilled physiologic salt solution (PSS) with a composition (mm) of 110.5 NaCl, 25.7 NaHCO3, 5.6 dextrose, 3.4 KCL, 2.4 CaCl2, 1.2 KH2PO4, and 0.8 mg SO4bubbled with 94% and 6% CO2(pH ∼ 7.4). After removal of fat, connective tissue, and epithelium, two sizes of smooth muscle strips were prepared: 1.5 mm wide × 10 mm long for organ bath studies and 0.5 mm wide × 10 mm long for superfusion studies. Strips in the organ baths were used to determine concentration–response relationships, whereas simultaneous measurement of isometric force and [Ca2+]iwas performed in a superfusion apparatus.
Organ Bath Studies.
Tracheal strips were incubated in 4-ml organ baths filled with PSS (pH ∼ 7.4) bubbled with 94% O2and 6% CO2at 37°C. One end of each strip was anchored to a metal hook at the bottom of the tissue bath; the other end was attached to a calibrated force transducer (model FT03D; Grass Instrument, Quincy, MA). The strips were stretched between repeated contractions with 1 μm acetylcholine until isometric force was maximal (optimal length).
Superfusion Studies.
Tracheal strips were incubated in 10 ml of PSS (bubbled with 94% O2and 6% CO2at 22°C) containing the membrane-permeant acetoxy-methyl ester of fura-2 (fura-2/AM; 5 μm) for 3 h. Fura-2/AM was dissolved in dimethyl sulfoxide and 0.02% cremophor. After fura-2/AM loading, the strips were mounted in a 0.1-ml quartz cuvette and continuously superfused at 2 ml/min with PSS at 37°C (bubbled with 94% O2and 6% CO2) for 30 min to remove excess fura-2/AM. One end of the strips was anchored via  microforceps to a micrometer, and the other end was anchored via  microforceps to an isometric force transducer (model KG4; Scientific Instruments, Heidelberg, DE). During a 2-h equilibration period, the strips were stretched between repeated contractions with 1 μm acetylcholine until optimal length was achieved.
Measurement of [Ca2+]i
Fura-2 fluorescence intensity was measured with a photometric system that measures optical and mechanical parameters of isolated tissue simultaneously. Details of our system have been described elsewhere. 6 Light from a xenon high-pressure lamp was monochromatically filtered to restrict excitation light to 340-nm and 380-nm wavelengths. Fluorescence emitted from the strips was filtered at 500 ± 5 nm and detected by a photomultiplier assembly (Scientific Instruments). The emission fluorescence intensities due to excitation at 340-nm (F340) and 380-nm (F380) wavelengths were measured, and the F340to F380ratio (F340:F380) was used as an index of [Ca2+]i.
The effect of hexanol, which was used in superfusion studies as a representative primary alcohol, on fluorescence was checked with use of 10 mm hexanol, fura-2 pentapotassium salt, and solutions of varying free-Ca2+concentrations prepared with the Fabiato algorithm. 17 The effect of hexanol on autofluorescence of ASM strips was also checked with 10 mm hexanol. These studies showed that 10 mm hexanol had no effect on fluorescence of fura-2 or autofluorescence of ASM strips (data not shown).
Experimental Protocols
In these protocols, we first determined concentration-dependent effects of three primary alcohols (butanol, hexanol, and octanol) on isometric force produced by strips stimulated with either isotonic KCl or acetylcholine, which causes contraction of ASM by different means and thus provides different mechanistic information. KCl produces contraction by increasing [Ca2+]ivia  voltage-dependent Ca2+channels (VDCCs), whereas acetylcholine activates G-protein–coupled responses such as increases in Ca2+sensitivity and Ca2+entry via  VDCCs and receptor-operated calcium channels (also referred to as metabotropic cation channels). 18 These stimuli were applied at concentrations producing both maximal and half-maximal responses, because the effectiveness of contractile antagonists in ASM in general depends on the level of stimulation, a phenomenon known as functional antagonism. 19 We then chose to study hexanol to examine the effects of alcohols on [Ca2+]iand Ca2+sensitivity in two further protocols, because (1) its anesthetic potency is relatively similar to that of volatile anesthetics when expressed as concentration in aqueous solution (MAC value of ∼ 0.76 mm for hexanol, compared with ∼ 0.25 mm for halothane) 20 and (2) we had technical problems with longer-chain alcohols adsorbing to the plastic tubing used in the superfusion apparatus (see discussion in Materials).
Concentration-dependent Effects of Primary Alcohols on Isometric Force.
For each experiment, four strips from one dog were mounted in organ baths. After determination of optimal length, they were stimulated with either isotonic KCl (two sets of four strips) or acetylcholine (two sets of four strips). For each method of stimulation, a maximal response was first determined with use of 40 mm KCl or 10 μm acetylcholine. After washout, the strips were exposed either to maximal stimulation again or to a concentration of KCl or acetylcholine that produced approximately 50% of the maximal response, as determined individually for each strip. The concentrations necessary to produce this half-maximal response ranged from 20 to 28 mm for KCl and 0.1 to 0.3 μm for acetylcholine. After stable contractions were achieved, the strips were exposed to increasing concentrations of butanol, hexanol, or octanol. The fourth strip in each experiment was not exposed to an alcohol and served as a control for the effects of time.
Effects of Hexanol on Force and [Ca2+]i.
For each experiment, the strips were contracted with either isotonic KCl solution (40 mm) or acetylcholine (10 μm), concentrations which produce maximal contraction. After stable contractions were achieved (requiring ∼ 10 min), increasing concentrations of hexanol (1, 3, and 10 mm) were applied, and changes in F340:F380and force were recorded.
Effects of Hexanol on the Relationship between Force and [Ca2+]i.
A protocol similar to that used in our previous work was employed. 6 In two separate experiments, the effect of hexanol on this relationship was determined during stimulation with either 40 mm KCl (i.e.  , in the absence of muscarinic receptor stimulation) or 10 μm acetylcholine (i.e.  , in the presence of maximal muscarinic stimulation).
For acetylcholine experiments, the strips were first superfused with nominally Ca2+-free PSS containing 2 mm EGTA until both F340:F380and force were stable. The strips were stimulated with acetylcholine (10 μm) in Ca2+-free PSS containing 2 mm EGTA for 5 min, followed by Ca2+-free PSS containing 75 μm EGTA and 10 μm acetylcholine for 2 min. This produces a small, transient increase in [Ca2+]iand force as Ca2+is released from intracellular stores. Preliminary experiments and prior work 6 showed that this regimen (referred to hereafter as Ca2+depletion) was sufficient to completely suppress further Ca2+release from intracellular stores, with subsequent acetylcholine stimulation. The strips were then exposed to PSS containing 2.4 mm CaCl2, 75 μm EGTA, and 10 μm acetylcholine to define a maximal Ca2+response. After washout with Ca2+-free PSS containing 2 mm EGTA, the Ca2+depletion regimen was again performed. The cumulative concentration–response to CaCl2(0.05, 0.1, 0.2, 0.3, 0.5, 1.0, and 2.4 mm final concentrations) in the presence of 75 μm EGTA and 10 μm acetylcholine was then determined, thus defining a control relation between F340:F380and force in the presence of muscarinic stimulation. After washout with Ca2+-free PSS containing 2 mm EGTA, the Ca2+depletion regimen was again performed, followed by exposure to Ca2+-free PSS containing 75 μm EGTA and 10 μm acetylcholine. After 5 min, hexanol (5.5 mm) was then added to the solutions. This concentration was chosen on the basis of preliminary data showing that it causes a substantial but not maximal effect on both F340:F380and force during subsequent stimulation. After 5 min of hexanol exposure, the strips were exposed to 1.0 mm CaCl2(still containing hexanol and 10 μm acetylcholine) to elicit a contraction. After a stable response was achieved, the strips were exposed to hexanol-free solution containing 1.0 mm CaCl2(and 10 μm acetylcholine), which caused an immediate increase in force and F340:F380as the effects of hexanol dissipated. This maneuver permitted assessment of whether the effect of hexanol was fully reversible and whether the force–Ca2+relation was stable over the period of study.
For KCl stimulation a similar protocol was followed, except that the strips were exposed to 40 mm isotonic KCl instead of acetylcholine for the initial contraction, during the CaCl2concentration–response determination, and during and after hexanol exposure. Hexanol (2 mm) was used during KCl stimulation, and the concentration again was chosen on the basis of its providing a substantial but not maximal effect on F340:F380and force under these conditions. In addition, we found in preliminary experiments that the force–F340:F380relationship was not reproducible if high concentrations of CaCl2were used. Thus, the CaCl2concentrations used to determine the control force–F340:F380relationship were 0.08, 0.1, 0.2, 0.3, 0.5, and 0.8 mm CaCl2, and initial contraction with 40 mm KCl and the effect of hexanol were determined during exposure to 0.8 mm CaCl2.
Materials
Fura-2 was purchased from the manufacturer (Molecular Probes, Eugene, OR), as were all other drugs and chemicals used in the study (Sigma Chemical, St. Louis, MO). In organ bath studies, stock solutions of butanol, hexanol, and octanol were prepared with use of ethanol to provide appropriate dilutions. The final concentration of ethanol in organ baths was always less than 10 mm, a concentration which does not affect ASM force (data not shown). In superfusion system studies, hexanol was dissolved in PSS directly.
In preliminary studies, we measured the concentrations of alcohols in PSS to confirm that calculated concentrations were actually achieved in solution and were not limited by aqueous solubility. Concentrations were determined by gas chromatography with an electron capture detector (model 5880A; Hewlett-Packard, Waltham, MA), according to a modification of the method of Van Dyke and Wood. 21 In each case, we confirmed that the calculated concentration of alcohol was achieved in the organ bath up to the maximum concentration of each alcohol studied and that this concentration was stable over the period of study. We also measured concentrations of alcohols achieved in the superfusion system, finding that in the case of octanol, its concentration was significantly lower in solutions after passing through the system, a circumstance presumably reflecting adsorption into plastic tubing. This did not occur with hexanol.
Statistical Analysis
Data are expressed as mean ± SD; n represents the number of dogs. For concentration–response curves with force or F340:F380, values are expressed as a percentage of initial values before the addition of alcohols. Parameters for concentration–response curves were determined with use of nonlinear regression analysis fitting to a three-parameter Hill equation (Sigma Stat; Jandel Scientific, San Rafael, CA) and compared by means of unpaired t  tests.
For experiments determining the force–F340:F380relationship values were expressed as a percentage of the initial response to Ca2+(2.4 or 0.8 mm CaCl2for acetylcholine or KCl stimulation, respectively). To determine whether hexanol affected the force developed for a given [Ca2+]i(as measured by F340:F380), the F380:F340achieved during exposure to hexanol was determined for each experiment. The force associated with this F340:F380under control conditions (in the absence of hexanol) was calculated from the fitted Hill equation for that experiment. This force was compared with the actual force achieved during hexanol exposure by a paired t  test. A significant difference indicated that hexanol affected the force–F340:F380relation. A similar procedure was followed for the data obtained after hexanol washout to determine if the force–F340:F380relation had recovered. P  values of less than 0.05 were considered to be significant.
Results
Concentration-dependent Effects of Primary Alcohols on Isometric Force
In the absence of alcohol exposure, responses to both KCl and acetylcholine were stable over the course of the study (data not shown). Butanol, hexanol, and octanol decreased isometric force in a concentration-dependent manner during both KCl and acetylcholine-mediated contractions (figs. 1 and 2). Their potency increased as chain length increased, as indicated by a decrease in median effective concentration (EC50) values with chain length (table 1). Median effective concentration values were significantly higher during maximal stimulation than with half-maximal stimulation for both acetylcholine and KCl, indicative of functional antagonism. However, at high concentrations, each alcohol produced complete relaxation, even during maximal stimulation. When washed out of the strips, isometric force fully recovered for each alcohol studied (data not shown).
Fig. 1. Effects of primary alcohols on isometric force during stimulation with concentrations of isotonic KCl producing (A  ) maximal and (B  ) approximately half-maximal contractions. Circles, triangles, and squares represent octanol, hexanol, and butanol, respectively. Values are mean ± SD and represent a percentage of the initial force (n = 5 for each alcohol).
Fig. 1. Effects of primary alcohols on isometric force during stimulation with concentrations of isotonic KCl producing (A 
	) maximal and (B 
	) approximately half-maximal contractions. Circles, triangles, and squares represent octanol, hexanol, and butanol, respectively. Values are mean ± SD and represent a percentage of the initial force (n = 5 for each alcohol).
Fig. 1. Effects of primary alcohols on isometric force during stimulation with concentrations of isotonic KCl producing (A  ) maximal and (B  ) approximately half-maximal contractions. Circles, triangles, and squares represent octanol, hexanol, and butanol, respectively. Values are mean ± SD and represent a percentage of the initial force (n = 5 for each alcohol).
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Fig. 2. Effects of primary alcohols on isometric force during stimulation with concentrations of acetylcholine (ACh) producing (A  ) maximal and (B)  approximately half-maximal contractions. Circles, triangles, and squares represent octanol, hexanol, and butanol, respectively. Values are mean ± SD and represent a percentage of the initial force (n = 5 for each alcohol).
Fig. 2. Effects of primary alcohols on isometric force during stimulation with concentrations of acetylcholine (ACh) producing (A 
	) maximal and (B) 
	approximately half-maximal contractions. Circles, triangles, and squares represent octanol, hexanol, and butanol, respectively. Values are mean ± SD and represent a percentage of the initial force (n = 5 for each alcohol).
Fig. 2. Effects of primary alcohols on isometric force during stimulation with concentrations of acetylcholine (ACh) producing (A  ) maximal and (B)  approximately half-maximal contractions. Circles, triangles, and squares represent octanol, hexanol, and butanol, respectively. Values are mean ± SD and represent a percentage of the initial force (n = 5 for each alcohol).
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Table 1. Potency of Primary Alcohols
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Table 1. Potency of Primary Alcohols
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Effects of Hexanol on Force and [Ca2+]i
The application of hexanol produced a concentration-dependent decrease in both isometric force and [Ca2+]iduring maximal stimulation with either 40 mm KCl or 10 μm acetylcholine (Fig. 3). In both conditions, 10 mm hexanol caused almost complete relaxation. When hexanol was washed out, isometric force and [Ca2+]ifully recovered (data not shown).
Fig. 3. Effects of hexanol (A  ) on isometric force and (B  ) F340:F380(an index of intracellular [Ca2+]i) in canine airway smooth muscle contracted with 10 μm acetylcholine (closed circles, n = 6) or 40 mm KCl (open circles, n = 5). Values are mean ± SD and represent a percentage of the initial value before exposure to hexanol.
Fig. 3. Effects of hexanol (A 
	) on isometric force and (B 
	) F340:F380(an index of intracellular [Ca2+]i) in canine airway smooth muscle contracted with 10 μm acetylcholine (closed circles, n = 6) or 40 mm KCl (open circles, n = 5). Values are mean ± SD and represent a percentage of the initial value before exposure to hexanol.
Fig. 3. Effects of hexanol (A  ) on isometric force and (B  ) F340:F380(an index of intracellular [Ca2+]i) in canine airway smooth muscle contracted with 10 μm acetylcholine (closed circles, n = 6) or 40 mm KCl (open circles, n = 5). Values are mean ± SD and represent a percentage of the initial value before exposure to hexanol.
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Effects of Hexanol on Relation between Force and [Ca2+]i
Figure 4shows a representative recording of the changes in force and [Ca2+]iinduced by the cumulative addition of CaCl2in the presence of acetylcholine and the effect of hexanol on force and [Ca2+]i. First, intracellular Ca2+stores were depleted by acetylcholine exposure, which caused a small transient increase in F340:F380and force. The response to 2.4 mm CaCl2was then determined. The cumulative addition of CaCl2to the Ca2+-free PSS containing 10 μm acetylcholine and 75 μm EGTA caused concentration-dependent increases in both force and F340:F380. During subsequent stimulation in the presence of 10 μm acetylcholine, hexanol (5.5 mm) inhibited increases in both force and [Ca2+]iproduced by 1.0 mm CaCl2(fig. 5). When hexanol was washed out of the strips, both force and F340:F380immediately increased to values similar to those measured during the initial determination of the force–F340:F380relationship.
Fig. 4. Representative tracing showing results from the protocol used to determine effect of hexanol on the relation between force and intracellular [Ca2+]iin the presence of muscarinic stimulation provided by acetylcholine. The upper panel shows changes in F340:F380(an index of intracellular [Ca2+]i), and the lower panel shows isometric force. After depletion of internal calcium stores and determination of the response to incremental increases in CaCl2, the effect of hexanol is determined. The dashed lines show that the response to 1 mm CaCl2after hexanol washout was similar to that measured before hexanol exposure.
Fig. 4. Representative tracing showing results from the protocol used to determine effect of hexanol on the relation between force and intracellular [Ca2+]iin the presence of muscarinic stimulation provided by acetylcholine. The upper panel shows changes in F340:F380(an index of intracellular [Ca2+]i), and the lower panel shows isometric force. After depletion of internal calcium stores and determination of the response to incremental increases in CaCl2, the effect of hexanol is determined. The dashed lines show that the response to 1 mm CaCl2after hexanol washout was similar to that measured before hexanol exposure.
Fig. 4. Representative tracing showing results from the protocol used to determine effect of hexanol on the relation between force and intracellular [Ca2+]iin the presence of muscarinic stimulation provided by acetylcholine. The upper panel shows changes in F340:F380(an index of intracellular [Ca2+]i), and the lower panel shows isometric force. After depletion of internal calcium stores and determination of the response to incremental increases in CaCl2, the effect of hexanol is determined. The dashed lines show that the response to 1 mm CaCl2after hexanol washout was similar to that measured before hexanol exposure.
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Fig. 5. The relationship between [CaCl2] and (A  ) force or (B  ) F340:F380in the presence of 10 μm acetylcholine (ACh), according to the protocol indicated in figure 4. Closed circles indicate values measured during the initial exposure to CaCl2, open circles indicate values measured in the presence of 5.5 mm hexanol, and gray circles indicate values after washout of hexanol. Values are mean ± SD, expressed as a percentage of the initial response to 2.4 mm CaCl2(n = 5).
Fig. 5. The relationship between [CaCl2] and (A 
	) force or (B 
	) F340:F380in the presence of 10 μm acetylcholine (ACh), according to the protocol indicated in figure 4. Closed circles indicate values measured during the initial exposure to CaCl2, open circles indicate values measured in the presence of 5.5 mm hexanol, and gray circles indicate values after washout of hexanol. Values are mean ± SD, expressed as a percentage of the initial response to 2.4 mm CaCl2(n = 5).
Fig. 5. The relationship between [CaCl2] and (A  ) force or (B  ) F340:F380in the presence of 10 μm acetylcholine (ACh), according to the protocol indicated in figure 4. Closed circles indicate values measured during the initial exposure to CaCl2, open circles indicate values measured in the presence of 5.5 mm hexanol, and gray circles indicate values after washout of hexanol. Values are mean ± SD, expressed as a percentage of the initial response to 2.4 mm CaCl2(n = 5).
×
For the mean data, there was a sigmoidal relationship between force or F340:F380and [CaCl2] during the initial control determination (fig. 5). Hexanol decreased both the force and the F340:F380produced by 1.0 mm CaCl2. When hexanol was washed out, both the force and F340:F380produced by 1.0 mm CaCl2approximated control values. When the data were expressed as the relationship between force and F340:F380by the pairing of data at common values of [CaCl2], it was apparent that hexanol changed this relationship such that the force produced by a given F340:F380was decreased in the presence of hexanol (fig. 6A). Indeed, there was a significant difference between the mean value of force obtained in the presence of hexanol and the value of the force predicted from the control relationship at the same F340:F380(21 ± 8% and 49 ± 12%, respectively;P  < 0.0001). Thus, during muscarinic stimulation, hexanol decreased Ca2+sensitivity, defined as the force maintained for a given [Ca2+]i. After hexanol washout, there was no significant difference between the mean value of force obtained after washout and the value of the force predicted from the control relation at the same F340:F380(100 ± 28% and 106 ± 17%, respectively;P  = 0.5). This indicated that the effects of hexanol were completely reversible and that the force–[Ca2+]irelation was stable over the period of study.
Fig. 6. The relationship between F340:F380and force during exposure to (A  , n = 5) 10 μm acetylcholine (ACh) or (B  , n = 5) 40 mm KCl, plotted from the data presented in figures 5 and 7. Closed circles indicate values measured during the initial exposure to CaCl2, open circles indicate values measured in the presence of hexanol (5.5 and 2 mm for ACh and KCl-exposed strips, respectively), and gray circles indicate values after washout of hexanol. Values are mean ± SD, expressed as a percentage of the initial response to CaCl2(2.4 mm and 0.8 mm for ACh and KCl-exposed strips, respectively).
Fig. 6. The relationship between F340:F380and force during exposure to (A 
	, n = 5) 10 μm acetylcholine (ACh) or (B 
	, n = 5) 40 mm KCl, plotted from the data presented in figures 5 and 7. Closed circles indicate values measured during the initial exposure to CaCl2, open circles indicate values measured in the presence of hexanol (5.5 and 2 mm for ACh and KCl-exposed strips, respectively), and gray circles indicate values after washout of hexanol. Values are mean ± SD, expressed as a percentage of the initial response to CaCl2(2.4 mm and 0.8 mm for ACh and KCl-exposed strips, respectively).
Fig. 6. The relationship between F340:F380and force during exposure to (A  , n = 5) 10 μm acetylcholine (ACh) or (B  , n = 5) 40 mm KCl, plotted from the data presented in figures 5 and 7. Closed circles indicate values measured during the initial exposure to CaCl2, open circles indicate values measured in the presence of hexanol (5.5 and 2 mm for ACh and KCl-exposed strips, respectively), and gray circles indicate values after washout of hexanol. Values are mean ± SD, expressed as a percentage of the initial response to CaCl2(2.4 mm and 0.8 mm for ACh and KCl-exposed strips, respectively).
×
The cumulative addition of CaCl2to the Ca2+-free PSS containing KCl and 75 μm EGTA also caused concentration-dependent increases in both force and F340:F380(fig. 6B). As during acetylcholine exposure, hexanol (2 mm) inhibited increases in both force and [Ca2+]i(fig. 7). However, in contrast to acetylcholine stimulation, hexanol did not affect the relationship between force and F340:F380(fig. 6B). There was no significant difference between the mean values of force obtained in the presence of hexanol and the values of the force predicted from the control relation at the same F340:F380(13 ± 2%vs.  16 ± 8%). Thus, in the absence of receptor stimulation, hexanol relaxed the ASM exclusively by decreasing [Ca2+]i, without affecting Ca2+sensitivity. As with acetylcholine stimulation, after hexanol washout there was no significant difference between the mean value of force obtained after washout and the value of the force predicted from the control relation at the same F340:F380(90 ± 9% and 82 ± 14%, respectively;P  = 0.2;fig. 6B).
Fig. 7. The relationship between [CaCl2] and (A  ) force or (B  ) F340:F380in the presence of 40 mm KCl, according to the protocol indicated in figure 4. Closed circles indicate values measured during the initial exposure to CaCl2, open circles indicate values measured in the presence of 2 mm hexanol, and gray circles indicate values after washout of hexanol. Values are mean ± SD, expressed as a percentage of the initial response to 0.8 mm CaCl2(n = 5).
Fig. 7. The relationship between [CaCl2] and (A 
	) force or (B 
	) F340:F380in the presence of 40 mm KCl, according to the protocol indicated in figure 4. Closed circles indicate values measured during the initial exposure to CaCl2, open circles indicate values measured in the presence of 2 mm hexanol, and gray circles indicate values after washout of hexanol. Values are mean ± SD, expressed as a percentage of the initial response to 0.8 mm CaCl2(n = 5).
Fig. 7. The relationship between [CaCl2] and (A  ) force or (B  ) F340:F380in the presence of 40 mm KCl, according to the protocol indicated in figure 4. Closed circles indicate values measured during the initial exposure to CaCl2, open circles indicate values measured in the presence of 2 mm hexanol, and gray circles indicate values after washout of hexanol. Values are mean ± SD, expressed as a percentage of the initial response to 0.8 mm CaCl2(n = 5).
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Discussion
The major findings of this study are that (1) the primary alcohols produce reversible, complete relaxation of ASM, with potency increasing as chain-length increases, and (2) like the volatile anesthetics, hexanol relaxes ASM both by decreasing [Ca2+]iand by inhibiting increases in Ca2+sensitivity produced by muscarinic receptor stimulation.
Previous Studies of Primary Alcohols and Smooth Muscle
Most previous studies of the effects of primary alcohols on smooth muscle have concerned ethanol. In general, ethanol relaxes smooth muscle, although contraction has been noted in some settings. 22 In previous work we found that ethanol also relaxes ASM contracted with acetylcholine. 13 However, the maximal relaxation produced by ethanol in that study was limited to approximately 40%. In contrast, the primary alcohols examined in the current study could produce complete ASM relaxation. There have been no studies of the effects of longer-chain primary alcohols on ASM. Rang 23 found that longer-chain primary alcohols depressed acetylcholine-induced contraction of guinea-pig ileum, with EC50values for this effect somewhat higher than those found in the current study (values of 27, 2.5, and 0.25 mm for butanol, hexanol, and octanol, respectively).
Effects on [Ca2+]i
We studied the effects of primary alcohols on contractions elicited by both KCl and acetylcholine, which produce sustained increases in [Ca2+]iby somewhat different mechanisms. The ability of the alcohols to decrease force during KCl stimulation implies that they inhibit Ca2+influx via  VDCCs activated by membrane depolarization. Indeed, hexanol produced a concentration-dependent decrease in [Ca2+]i. This finding is consistent with the effects of primary alcohols on Ca2+channels in a variety of tissues. 24–26 Data pertaining specifically to smooth muscle are more limited but suggest that ethanol reduces [Ca2+]i, possibly by affecting VDCCs. 27,28 Volatile anesthetics have similar effects on VDCCs in a variety of tissues, including ASM. 4–7,29,30 
Acetylcholine also produces Ca2+influx via  VDCCs, but in addition it activates receptor-operated calcium channels coupled to muscarinic receptors and causes the release of Ca2+from stores in the sarcoplasmic reticulum. Receptor-operated calcium channels are an especially important pathway for Ca2+influx during maximal muscarinic stimulation of ASM. 30 The ability of hexanol to completely eliminate acetylcholine-induced increases in [Ca2+]i, even during maximal muscarinic stimulation, implies an effect on receptor-operated calcium channels. Hexanol also may affect Ca2+release from the sarcoplasmic reticulum, although this possibility was not evaluated in the present study. The apparent effect of hexanol on receptor-operated calcium channels contrasts with the findings of a previous study in which halothane (0.6 mm) did not affect [Ca2+]ior Ca2+influx in ASM during maximal muscarinic stimulation. 30 This discrepancy may reflect differences in the relative concentrations of halothane and hexanol studied, although the comparable concentration of hexanol in terms of MAC equivalents in aqueous solution (∼ 2 mm hexanol) did reduce [Ca2+]iin the current study (fig. 3B).
We believe that the ability of hexanol to reversibly reduce [Ca2+]iduring maximal muscarinic stimulation—an ability presumably shared by the other primary alcohols studied, which also could completely relax ASM under this condition—is unique. In general, the ability of bronchodilators to relax ASM diminishes as the level of muscarinic stimulation increases, a phenomenon known as functional antagonism. 19 For example, neither antagonists of VDCCs (e.g.  , verapamil) nor β-adrenergic agonists (e.g.  , isoproterenol) produce appreciable relaxation when applied to ASM during maximal muscarinic stimulation. 19,30 Evidence of functional antagonism in response to the primary alcohols is also seen in our findings, as the EC50values for each alcohol were significantly greater for maximal stimulation with both KCl and acetylcholine. However, higher alcohol concentrations could overcome this effect and produce full relaxation.
Effects on Ca2+Sensitivity
Other mechanisms control force produced by ASM independently of changes in [Ca2+]i. These mechanisms are activated by stimulation of muscarinic and other membrane receptors. We assessed changes in Ca2+sensitivity by determining the relation between force and F340:F380, an index of [Ca2+]i, before and after exposure to hexanol. Although permeabilized ASM preparations permit more detailed exploration of mechanism, this technique has the advantage of enabling the study of intact ASM at physiologic temperatures. The effects of hexanol depended on the presence or absence of muscarinic receptor stimulation. In the absence of receptor stimulation (with Ca2+entry permitted by KCl-induced membrane polarization), hexanol (2 mm) decreased force solely by decreasing [Ca2+]i, with no change in the force–Ca2+relationship (fig. 6B). This finding suggests that hexanol (2 mm) does not affect elements of the contractile system such as calmodulin, myosin light chain kinase, or myosin and is similar to that reported for halothane. 6,29 During muscarinic receptor stimulation, hexanol decreased force both by decreasing [Ca2+]iand by decreasing the force produced for a given [Ca2+]i(fig. 6A). Taken with the results obtained during KCl depolarization, this finding suggests that hexanol affects receptor-linked second messenger systems such as G-proteins that increase Ca2+sensitivity during muscarinic receptor stimulation. We have shown a similar pattern of results for halothane under similar conditions. 6,29 Because it was necessary to use a higher concentration of hexanol during muscarinic stimulation (5.5 mm), we cannot exclude that this higher concentration of hexanol might have also decreased Ca2+sensitivity in the absence of receptor stimulation.
In a previous study in permeabilized ASM, we found that ethanol in a high concentration (240 mm) also affected receptor-linked control of Ca2+sensitivity, causing a decrease in regulatory myosin light chain phosphorylation during muscarinic receptor stimulation. 13 However, this action, which favors decreases in Ca2+sensitivity, was offset by a concurrent effect of ethanol to increase Ca2+sensitivity in the absence of receptor stimulation. This occurred by a mechanism independent of changes in regulatory myosin light chain phosphorylation. The net result of these two actions was that ethanol actually increased Ca2+sensitivity. In the current study, we found no evidence of this latter mechanism in the action of hexanol, which had no effect on Ca2+sensitivity in the absence of receptor stimulation. This difference in the effects of ethanol and hexanol on Ca2+sensitivity may partially explain why the maximal ASM relaxation produced by ethanol is limited to approximately 40%, 13 whereas hexanol and the other primary alcohols studied can completely relax ASM.
Relation of Alcohol Potency in ASM and Anesthetic Effect
Minimum alveolar concentration values for anesthetic effects (expressed as concentration in saline solution with use of available solubility data) in the rat are approximately 7.3, 0.76, and 0.032 mm for butanol, hexanol, and octanol, respectively. 12 Relatively similar values are reported for the EC50for the righting reflex in tadpoles, although these may not be directly comparable because this is a somewhat different parameter measured at room temperature. 11 These findings suggest that the EC50values we noted for the effects of primary alcohols on ASM (table 1) are relevant to alcohol concentrations necessary to achieve anesthesia. Relative to anesthetizing concentrations, the primary alcohols may be even more potent bronchodilators than the volatile anesthetics. During half-maximal stimulation with acetylcholine, the relaxation produced by concentrations equivalent to 1 MAC in aqueous solution was approximately 60%, 80%, and 60% for butanol, hexanol, and octanol, respectively (fig. 2). In a previous study we found that 1 MAC halothane produces approximately 20–30% relaxation under similar conditions. 31–33 The concentration–response relationship for effects on ASM was steep, as is found for the anesthetic effects of alcohols (and other anesthetics). 12 The effect of chain length on anesthetic potency can be compared with its effect on the alcohol's potency to relax ASM by calculation of the ratio of EC50values (or MAC values, as appropriate) for pairs of alcohols. Although there is some variability, these potency ratios are quite similar for the effects on tadpole righting reflex 11 and ASM (table 2). They are less similar to MAC values in the rat 12 but still comparable. Thus, like volatile anesthetics, the direct effects of primary alcohols on ASM occur at concentrations that are relevant to anesthesia, and the concentration–response relations for effects on ASM and the anesthetic state share other common features.
Table 2. Potency Ratios for Effects on Airway Smooth Muscle and Anesthetic Effects
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Table 2. Potency Ratios for Effects on Airway Smooth Muscle and Anesthetic Effects
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The effects of primary alcohols on MAC are approximately additive with volatile anesthetics, which has prompted the speculation that their mechanisms of action have much in common. 12 On the other hand, the primary alcohols are approximately 10-fold more potent in producing anesthesia than predicted from their solubility in olive oil, and by this measure they do not obey the Meyer-Overton hypothesis, unlike volatile anesthetics. 12 It has been suggested that the OH moiety increases alcohol affinity to a site of anesthetic action, implying that such a site contains both polar and nonpolar domains.
Alcohols as Bronchodilators
Although the primary aim of this work was to investigate the primary alcohols as tools for studying anesthetic mechanisms of action in ASM, these results suggest that they or related compounds could possibly serve as bronchodilators. Their ability to completely relax ASM even when it is maximally stimulated would be particularly useful in the treatment of conditions such as status asthmaticus. Although data are limited, ethanol may decrease pulmonary resistance in human subjects when given by aerosol. 34,35 Ethanol aerosol has in fact been employed clinically as a bronchodilator and antifoaming agent in the treatment of pulmonary edema, with variable results. 36 As noted above, the inability of ethanol to completely relax ASM in a previous study 13 may mean that other alcohols would be more suitable for this purpose. Sedating effects of absorbed aerosolized alcohols may limit their application, although this would not be an issue if these alcohols were administered to patients in status asthmaticus receiving respiratory support. However, several issues, such as the possibility of chronic toxicity and the efficiency of absorption through the airway epithelial barrier, would need to be addressed before this approach could be recommended.
Summary
Primary alcohols produce reversible, complete relaxation of ASM, with potency increasing as chain length increases, by decreasing [Ca2+]iand inhibiting increases in Ca2+sensitivity produced by muscarinic receptor stimulation. These actions mimic those of volatile anesthetics on ASM in many respects, suggesting that the primary alcohols may be useful tools for further exploring mechanisms of anesthetic effects on ASM.
The authors thank the following persons at the Department of Anesthesiology of the Mayo Clinic (Rochester, MN): Kathy Street (Research Technician) and Darrell Loeffler (Research Technician) for expert technical assistance, Nicole Weber (Interim Technical Student) and Keri Griffin (Interim Technical Student) for assistance with the experiments, and Janet Beckman (Secretary) for secretarial support.
References
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Fig. 1. Effects of primary alcohols on isometric force during stimulation with concentrations of isotonic KCl producing (A  ) maximal and (B  ) approximately half-maximal contractions. Circles, triangles, and squares represent octanol, hexanol, and butanol, respectively. Values are mean ± SD and represent a percentage of the initial force (n = 5 for each alcohol).
Fig. 1. Effects of primary alcohols on isometric force during stimulation with concentrations of isotonic KCl producing (A 
	) maximal and (B 
	) approximately half-maximal contractions. Circles, triangles, and squares represent octanol, hexanol, and butanol, respectively. Values are mean ± SD and represent a percentage of the initial force (n = 5 for each alcohol).
Fig. 1. Effects of primary alcohols on isometric force during stimulation with concentrations of isotonic KCl producing (A  ) maximal and (B  ) approximately half-maximal contractions. Circles, triangles, and squares represent octanol, hexanol, and butanol, respectively. Values are mean ± SD and represent a percentage of the initial force (n = 5 for each alcohol).
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Fig. 2. Effects of primary alcohols on isometric force during stimulation with concentrations of acetylcholine (ACh) producing (A  ) maximal and (B)  approximately half-maximal contractions. Circles, triangles, and squares represent octanol, hexanol, and butanol, respectively. Values are mean ± SD and represent a percentage of the initial force (n = 5 for each alcohol).
Fig. 2. Effects of primary alcohols on isometric force during stimulation with concentrations of acetylcholine (ACh) producing (A 
	) maximal and (B) 
	approximately half-maximal contractions. Circles, triangles, and squares represent octanol, hexanol, and butanol, respectively. Values are mean ± SD and represent a percentage of the initial force (n = 5 for each alcohol).
Fig. 2. Effects of primary alcohols on isometric force during stimulation with concentrations of acetylcholine (ACh) producing (A  ) maximal and (B)  approximately half-maximal contractions. Circles, triangles, and squares represent octanol, hexanol, and butanol, respectively. Values are mean ± SD and represent a percentage of the initial force (n = 5 for each alcohol).
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Fig. 3. Effects of hexanol (A  ) on isometric force and (B  ) F340:F380(an index of intracellular [Ca2+]i) in canine airway smooth muscle contracted with 10 μm acetylcholine (closed circles, n = 6) or 40 mm KCl (open circles, n = 5). Values are mean ± SD and represent a percentage of the initial value before exposure to hexanol.
Fig. 3. Effects of hexanol (A 
	) on isometric force and (B 
	) F340:F380(an index of intracellular [Ca2+]i) in canine airway smooth muscle contracted with 10 μm acetylcholine (closed circles, n = 6) or 40 mm KCl (open circles, n = 5). Values are mean ± SD and represent a percentage of the initial value before exposure to hexanol.
Fig. 3. Effects of hexanol (A  ) on isometric force and (B  ) F340:F380(an index of intracellular [Ca2+]i) in canine airway smooth muscle contracted with 10 μm acetylcholine (closed circles, n = 6) or 40 mm KCl (open circles, n = 5). Values are mean ± SD and represent a percentage of the initial value before exposure to hexanol.
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Fig. 4. Representative tracing showing results from the protocol used to determine effect of hexanol on the relation between force and intracellular [Ca2+]iin the presence of muscarinic stimulation provided by acetylcholine. The upper panel shows changes in F340:F380(an index of intracellular [Ca2+]i), and the lower panel shows isometric force. After depletion of internal calcium stores and determination of the response to incremental increases in CaCl2, the effect of hexanol is determined. The dashed lines show that the response to 1 mm CaCl2after hexanol washout was similar to that measured before hexanol exposure.
Fig. 4. Representative tracing showing results from the protocol used to determine effect of hexanol on the relation between force and intracellular [Ca2+]iin the presence of muscarinic stimulation provided by acetylcholine. The upper panel shows changes in F340:F380(an index of intracellular [Ca2+]i), and the lower panel shows isometric force. After depletion of internal calcium stores and determination of the response to incremental increases in CaCl2, the effect of hexanol is determined. The dashed lines show that the response to 1 mm CaCl2after hexanol washout was similar to that measured before hexanol exposure.
Fig. 4. Representative tracing showing results from the protocol used to determine effect of hexanol on the relation between force and intracellular [Ca2+]iin the presence of muscarinic stimulation provided by acetylcholine. The upper panel shows changes in F340:F380(an index of intracellular [Ca2+]i), and the lower panel shows isometric force. After depletion of internal calcium stores and determination of the response to incremental increases in CaCl2, the effect of hexanol is determined. The dashed lines show that the response to 1 mm CaCl2after hexanol washout was similar to that measured before hexanol exposure.
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Fig. 5. The relationship between [CaCl2] and (A  ) force or (B  ) F340:F380in the presence of 10 μm acetylcholine (ACh), according to the protocol indicated in figure 4. Closed circles indicate values measured during the initial exposure to CaCl2, open circles indicate values measured in the presence of 5.5 mm hexanol, and gray circles indicate values after washout of hexanol. Values are mean ± SD, expressed as a percentage of the initial response to 2.4 mm CaCl2(n = 5).
Fig. 5. The relationship between [CaCl2] and (A 
	) force or (B 
	) F340:F380in the presence of 10 μm acetylcholine (ACh), according to the protocol indicated in figure 4. Closed circles indicate values measured during the initial exposure to CaCl2, open circles indicate values measured in the presence of 5.5 mm hexanol, and gray circles indicate values after washout of hexanol. Values are mean ± SD, expressed as a percentage of the initial response to 2.4 mm CaCl2(n = 5).
Fig. 5. The relationship between [CaCl2] and (A  ) force or (B  ) F340:F380in the presence of 10 μm acetylcholine (ACh), according to the protocol indicated in figure 4. Closed circles indicate values measured during the initial exposure to CaCl2, open circles indicate values measured in the presence of 5.5 mm hexanol, and gray circles indicate values after washout of hexanol. Values are mean ± SD, expressed as a percentage of the initial response to 2.4 mm CaCl2(n = 5).
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Fig. 6. The relationship between F340:F380and force during exposure to (A  , n = 5) 10 μm acetylcholine (ACh) or (B  , n = 5) 40 mm KCl, plotted from the data presented in figures 5 and 7. Closed circles indicate values measured during the initial exposure to CaCl2, open circles indicate values measured in the presence of hexanol (5.5 and 2 mm for ACh and KCl-exposed strips, respectively), and gray circles indicate values after washout of hexanol. Values are mean ± SD, expressed as a percentage of the initial response to CaCl2(2.4 mm and 0.8 mm for ACh and KCl-exposed strips, respectively).
Fig. 6. The relationship between F340:F380and force during exposure to (A 
	, n = 5) 10 μm acetylcholine (ACh) or (B 
	, n = 5) 40 mm KCl, plotted from the data presented in figures 5 and 7. Closed circles indicate values measured during the initial exposure to CaCl2, open circles indicate values measured in the presence of hexanol (5.5 and 2 mm for ACh and KCl-exposed strips, respectively), and gray circles indicate values after washout of hexanol. Values are mean ± SD, expressed as a percentage of the initial response to CaCl2(2.4 mm and 0.8 mm for ACh and KCl-exposed strips, respectively).
Fig. 6. The relationship between F340:F380and force during exposure to (A  , n = 5) 10 μm acetylcholine (ACh) or (B  , n = 5) 40 mm KCl, plotted from the data presented in figures 5 and 7. Closed circles indicate values measured during the initial exposure to CaCl2, open circles indicate values measured in the presence of hexanol (5.5 and 2 mm for ACh and KCl-exposed strips, respectively), and gray circles indicate values after washout of hexanol. Values are mean ± SD, expressed as a percentage of the initial response to CaCl2(2.4 mm and 0.8 mm for ACh and KCl-exposed strips, respectively).
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Fig. 7. The relationship between [CaCl2] and (A  ) force or (B  ) F340:F380in the presence of 40 mm KCl, according to the protocol indicated in figure 4. Closed circles indicate values measured during the initial exposure to CaCl2, open circles indicate values measured in the presence of 2 mm hexanol, and gray circles indicate values after washout of hexanol. Values are mean ± SD, expressed as a percentage of the initial response to 0.8 mm CaCl2(n = 5).
Fig. 7. The relationship between [CaCl2] and (A 
	) force or (B 
	) F340:F380in the presence of 40 mm KCl, according to the protocol indicated in figure 4. Closed circles indicate values measured during the initial exposure to CaCl2, open circles indicate values measured in the presence of 2 mm hexanol, and gray circles indicate values after washout of hexanol. Values are mean ± SD, expressed as a percentage of the initial response to 0.8 mm CaCl2(n = 5).
Fig. 7. The relationship between [CaCl2] and (A  ) force or (B  ) F340:F380in the presence of 40 mm KCl, according to the protocol indicated in figure 4. Closed circles indicate values measured during the initial exposure to CaCl2, open circles indicate values measured in the presence of 2 mm hexanol, and gray circles indicate values after washout of hexanol. Values are mean ± SD, expressed as a percentage of the initial response to 0.8 mm CaCl2(n = 5).
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Table 1. Potency of Primary Alcohols
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Table 1. Potency of Primary Alcohols
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Table 2. Potency Ratios for Effects on Airway Smooth Muscle and Anesthetic Effects
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Table 2. Potency Ratios for Effects on Airway Smooth Muscle and Anesthetic Effects
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