In airway smooth muscle, muscarinic receptor stimulation is thought to increase calcium (Ca2+) sensitivity via a guanosine 5'-triphosphate (GTP)-binding protein/protein kinase C (PKC)-mediated mechanism. This study treated the hypothesis that halothane reduces Ca2+ sensitivity during muscarinic receptor stimulation by inhibiting these second messenger pathways.
A beta-escin permeabilized canine tracheal smooth muscle preparation was used in which the cytosolic Ca2+ concentration ([Ca2+]i) is controlled and the GTP-binding protein/ PKC pathways remain intact and can be activated. The muscarinic receptor was activated with acetylcholine plus GTP; the GTP-binding proteins were directly activated with a nonhydrolyzable form of GTP, guanosine 5'-O-(3-thiotriphosphate; GTP gamma S); and PKC was directly activated with the PKC agonist phorbol 12,13-dibutyrate (PDBu).
Free Ca2+ caused a concentration-dependent increase in force. Acetylcholine plus GTP significantly decreased the median effective concentration for free Ca2+ from 0.52 +/- 0.06 microM to 0.21 +/- 0.02 microM, demonstrating an increase in Ca2+ sensitivity. Halothane (0.99 +/- 0.04 mM, equivalent to approximately 4 minimum alveolar concentration in dogs) significantly attenuated this increase in Ca2+ sensitivity induced by acetylcholine plus GTP, increasing the median effective concentration for free Ca2+ from 0.21 +/- 0.02 microM to 0.31 +/- 0.03 microM. However, halothane did not affect the increases in Ca2+ sensitivity induced by GTP gamma S or PDBu.
Halothane had no effect on increased Ca2+ sensitivity caused by direct activation of GTP-binding proteins with GTP gamma S or PKC with PDBu, suggesting that halothane attenuates acetylcholine-induced Ca2+ sensitization via a mechanism independent of these pathways in beta-escin-permeabilized canine tracheal smooth muscle.
Volatile anesthetics are potent bronchodilators, relaxing airway smooth muscle in vitro in part by a direct effect on the smooth muscle cell. [1–4] They reduce the cytosolic free calcium concentration ([Ca2+]i)[1–4] and the amount of force produced at a constant [Ca2+]i(i.e., calcium sensitivity [2,3]). Although the mechanisms by which volatile anesthetics decrease [Ca2+]ihave been investigated extensively, [1–4] few studies have elucidated the intracellular pathways by which these compounds reduce calcium sensitivity in airway smooth muscle,  a potentially important anesthetic mechanism.
Airway smooth muscle contraction produced by muscarinic receptor agonists is mediated by an increase in [Ca2+]i. Calcium binds calmodulin and subsequently increases myosin light chain kinase activity and phosphorylation of the 20-kDa regulatory myosin light chain. [6,7] Contractile force, however, does not depend on the increase in [Ca2+]ialone. Muscarinic receptor agonists also activate a guanosine 5'-triphosphate (GTP)-dependent second messenger cascade that increases Ca2+ sensitivity [8,9] in permeabilized smooth muscle preparations exposed to calcium. The mechanism of this action is not fully characterized, but there is considerable evidence that GTP-binding proteins [10–18] and protein kinase C (PKC)[19–22] play key roles.
The purpose of the current study was to determine the role of GTP-binding proteins and PKC as potential targets for halothane's action on acetylcholine-induced enhancement of Ca2+ sensitivity in canine tracheal smooth muscle (CTSM). We used a beta-escin-permeabilized CTSM preparation in which pores are produced in the sarcolemma, permitting control of [Ca2+]iby manipulation of extracellular Ca2+ concentration, yet GTP-binding protein and PKC second messenger cascades remain intact and can be activated. [5,23]
Materials and Methods
After we received approval from the Institutional Animal Care and Use Committee, and conforming to the Guiding Principles in the Care and Use of Animals as approved by the Council of the American Physiological Society, we anesthetized 33 mongrel dogs (15–25 kg) of either sex with an intravenous injection of pentobarbital (30 mg/kg) and then killed them by exsanguination. A 15- to 20-cm portion of extrathoracic trachea was excised and immersed in chilled physiologic salt solution of the following composition: 110.5 mM NaCl, 25.7 mM NaHCO3, 5.6 mM dextrose, 3.4 mM KCl, 2.4 mM CaCl2, 1.2 mM KH2PO4, and 0.8 mM MgSO4. Fat, connective tissue, and the epithelium were removed with tissue forceps and scissors under microscopic observation.
Isometric Force Measurements. Muscle strips (width, 0.1–0.2 mm; length, 3–5 mm; wet weight, 0.05–0.1 mg) were mounted in a 0.1-ml cuvette and continuously perfused at 2 ml/min with physiologic salt solution (at 37 [degree sign] Celsius) aerated with 94% oxygen and 6% carbon dioxide. One end of the strips was anchored with stainless steel microforceps to a stationary metal rod and the other end was anchored with stainless steel microforceps to a calibrated force transducer (model KG4; Scientific Instruments, Heidelberg, Germany). During a 3-h equilibration period, the length of the strips was increased after repeated isometric contractions (lasting 2 or 3 min) induced by 1 micro Meter acetylcholine until the optimal length was obtained. Each strip was maintained at this optimal length and cooled to room temperature (25 [degree sign] Celsius) for the rest of the experiment. These tissues produced isometric forces of 0.8–2.5 mN.
Permeabilization Procedure. Muscle strips were permeabilized with beta-escin as previously described [12,18,23] and validated in our laboratory.  beta-Escin creates pores in the sarcolemma, thus allowing substances of small molecular weight, such as Ca2+, to diffuse freely across the cell membrane. Accordingly, [Ca2+]ican be manipulated by changing the extracellular Ca2+ concentration with ethyleneglycol-bis-(beta-aminoethylether)-N,N,N'N'-tetraaectic acid (EGTA)-buffered solutions in the bathing media. Larger cellular proteins necessary for contraction are preserved and the membrane receptor-coupled second messenger systems thought to regulate Ca2+ sensitivity, such as GTP-binding proteins and PKC, remain intact and can be activated.  Thus effects of halothane on Ca2+-calmodulin activation of the contractile proteins can be distinguished from those on the membrane receptor-coupled second messenger systems that regulate Ca2+ sensitivity.
Muscle strips were permeabilized for 20 min with 100 micro Meter beta-escin in relaxing solution. The composition of the relaxing solution was 7.5 mM adenosine 5'-triphosphate, disodium salt; 4 mM EGTA; 20 mM imidazole; 1 mM dithiothreitol; 1 nM free Ca2+; 10 mM creatinine phosphate; and 0.1 mg/ml creatinine phosphokinase. The pH was buffered to 7.0 at 25 [degree sign] Celsius with potassium hydroxide; the ionic strength was kept constant at 0.20 M by adjusting the concentration of potassium acetate. After the permeabilization procedure, strips were perfused with relaxing solution for 10 min to wash out the excess beta-escin. Calcium ionophore A23187 (10 micro Meter) was added to the relaxing solution and all subsequent experimental solutions to deplete the sarcoplasmic reticulum Ca2+ stores. Solutions of various free Ca2+ concentrations were prepared using the algorithm of Fabiato and Fabiato.  In all experimental solutions, the final concentration of dimethyl sulfoxide, used as a vehicle to dissolve compounds insoluble in water, did not exceed 0.01%; at this concentration, dimethyl sulfoxide had no effect on the isometric force development (data not shown).
Experiments were conducted to determine the effects of halothane on Ca2+ sensitization induced by muscarinic receptor stimulation with 3 micro Meter acetylcholine plus 10 micro Meter GTP, by direct activation of GTP-binding proteins with a nonhydrolyzable analog of GTP, guanosine 5'-O-(3-thiotriphosphate)(GTP gamma S) and by direct activation of PKC with the phorbol ester phorbol 12, 13-dibutyrate (PDBu). Previously we found that halothane has no effect on Ca2+ sensitivity in the absence of muscarinic receptor stimulation,  indicating that halothane has no effect on Ca2+-calmodulin activation of myosin light chain kinase or the contractile proteins.
Two experimental protocols were performed. In the first protocol, the effect of agonist-induced Ca2+ sensitization in the absence and presence of halothane (0.99 +/- 0.04 mM, equivalent to approximately 4 minimum alveolar concentration for dogs expressed as an aqueous concentration in saline, corrected to room temperature ) was determined by generating free Ca2+ concentration-response curves (0.001–10 micro Meter). These experiments included three tissue sets, one for each agonist. In each tissue set, three permeabilized CTSM strips were prepared and studied concomitantly. One strip of each set was perfused with relaxing solution alone (Ca2+ alone, control). The other two strips of each set were perfused with relaxing solution containing either 3 micro Meter acetylcholine (a concentration causing 80% of its maximal effect on isometric force development at a constant [Ca2+]iin this preparation ) plus 10 micro Meter GTP to activate muscarinic receptors (n = 6), 3 micro Meter GTP gamma S (n = 6), or 1 micro Meter PDBu (n = 6). One strip was also exposed to halothane. To generate the Ca sup 2+ concentration-response curves, muscle strips were allowed to contract for 10 min after each increment of free Ca2+ concentration. After these determinations, strips were perfused with relaxing solution for 10 min, including inorganic phosphate (Pi; 5 mM) to reduce the time required for relaxation by accelerating the rate of cross-bridge detachment  and then perfused with relaxing solution for 10 min to remove Pi. Isometric force measurements were normalized to those determined in the same tissues in response to 10 micro Meter free Ca2+ at the end of the experiment.
The second experimental protocol was performed to determine the effect of halothane on different intensities of GTP-binding protein or PKC activation. Concentration-response curves for GTP gamma S and PDBu were generated at a constant submaximal [Ca2+]iof 0.1 micro Meter and 0.3 micro Meter, respectively. Preliminary studies showed that force developed in response to PDBu only in the presence of >or= to 0.1 micro Meter [Ca2+]i. Two pairs of two permeabilized CTSM strips were prepared and the contractile response to increasing concentrations of GTP gamma S (0.01–31.6 micro Meter; n = 5) or PDBu (0.001–3 micro Meter; n = 5) was measured. The solutions perfusing one strip of each pair also contained 0.97 +/- 0.13 mM halothane. Muscle strips were allowed to contract for 10 min after each increment of concentration, the time for stable contractile responses. Isometric force measurements were normalized to those determined in the same tissues in response to 10 micro Meter free Ca2+.
Administration of Halothane. Halothane was delivered to solutions via an on-line calibrated vaporizer. Each solution was equilibrated with halothane for at least 5 min before being introduced to the system. The concentrations of halothane in the solutions at the cuvette were determined by gas chromatography from anaerobically obtained samples using an electron capture detector (model 5880A; Hewlett-Packard, Waltham, MA) according to the method of Van Dyke and Wood. 
Materials. Halothane was purchased from Wyeth-Ayerst Laboratories, Inc. (Philadelphia, PA). A23187 was purchased from Molecular Probes, Inc. (Eugene, OR). Adenosine 5'-triphosphate, disodium salt, was purchased from Research Organics, Inc. (Cleveland, OH). All other drugs and chemicals were purchased from Sigma Chemical Company (St. Louis, MO). Stock solutions and all other drugs and chemicals were prepared in distilled water or dimethyl sulfoxide.
Statistical Analysis. Data are expressed as mean values +/- SD; n represents the number of dogs. All forces were expressed as a percentage of the response to 10 micro Meter free Ca2+ in that strip. Concentration-response curves were compared by nonlinear regression described by Meddings et al.  In this method, force (F) at any concentration of drug C was given by the equation:Equation 1where F sub m represents the maximal isometric force generated under that condition and EC50represents the concentration that produces one half of this maximal isometric force for that drug. Nonlinear regression analysis was used to fit values of Fmand EC50to data for F and C for each condition studied. This method allowed us to compare curves to determine whether they were significantly different and whether this overall difference could be attributed to differences in Fm, EC50, or both parameters. Probability values of 0.05 or less were considered significant.
Effect of Halothane on Free Ca sup 2+ Concentration Response Curves during Muscarinic Receptor Stimulation
Ca2+ alone caused a concentration-dependent increase in force. Muscarinic receptor stimulation with acetylcholine plus GTP caused a leftward shift of the free Ca2+ concentration response curves (Figure 1), producing a significant additional force at a constant submaximal [Ca2+]ias compared to the force induced by free Ca sup 2+ alone (i.e., increasing Ca2+ sensitivity). The concentration of free Ca2+ required to produce 50% of maximal force (EC50) was significantly decreased by acetylcholine plus GTP from 0.52 +/- 0.06 micro Meter to 0.21 +/- 0.02 micro Meter. In the presence of acetylcholine and GTP, halothane caused a rightward shift of the free Ca2+ concentration-response curve, significantly increasing the EC50for free Ca2+ from 0.21 +/- 0.02 micro Meter to 0.31 +/- 0.03 micro Meter.
Effect of Halothane on Ca sup 2+ Sensitization Induced by Guanosine 5'-O-(3-thiotriphosphate) or Phorbol 12, 13-dibutyrate
The possible involvement of GTP-binding proteins and PKC in halothane's attenuation of acetylcholine-induced Ca2+ sensitization was determined during activation with GTP gamma S and PDBu, respectively. Both GTP gamma S (Figure 2(A)) and PDBu (Figure 2(B)) caused a significant leftward shift of the free Ca2+ concentration-response curves, reducing the EC50for free Ca2+ from 0.61 +/- 0.09 micro Meter to 0.16 +/- 0.01 micro Meter and from 0.87 +/- 0.09 micro Meter to 0.29 +/- 0.04 micro Meter, respectively. In contrast to its effect on acetylcholine-induced Ca2+ sensitization, halothane had no effect on this leftward shift of the curves (GTP gamma S: 0.16 +/- 0.01 micro Meter to 0.18 +/- 0.02 micro Meter; PDBu: 0.29 +/- 0.04 micro Meter to 0.29 +/- 0.04 micro Meter, respectively).
GTP gamma S (Figure 3(A)) and PDBu (Figure 3(B)) increased force at a constant [Ca2+]iof 0.1 micro Meter or 0.3 micro Meter free Ca2+, respectively, in a concentration-dependent manner (i.e., enhanced Ca2+ sensitivity). The EC50for GTP gamma S was 1.17 +/- 0.31 micro Meter and was 0.21 +/- 0.02 micro Meter for PDBu. Halothane had no effect on this increase in Ca2+ sensitivity over the entire range of the agonist concentrations studied (EC50values of 1.46 +/- 0.25 micro Meter for GTP gamma S and 0.18 +/- 0.07 micro Meter for PDBu).
To confirm the involvement of GTP-binding proteins in acetylcholine-induced Ca2+ sensitization, the effect of guanosine 5'-O-(2-thiodiphosphate)(GDP beta S), a nonhydrolyzable form of GDP that antagonizes GTP-binding protein activation, was studied (n = 2). One mM GDP beta S had no effect on force induced by Ca2+ alone. Preincubation with 1 mM GDP beta S in relaxing solution inhibited subsequent force development induced by muscarinic receptor stimulation with 3 micro Meter acetylcholine and 10 micro Meter GTP in the presence of 0.3 micro Meter [Ca2+]iby approximately 90%. One mM GDP beta S added after muscarinic receptor stimulation caused an approximately 65% relaxation of the force induced by 3 micro Meter acetylcholine and 10 micro Meter GTP at 0.3 micro Meter [Ca2+]i(Figure 4).
The major findings of this study were that (1) halothane attenuated the potentiation in Ca2+ sensitivity caused by acetylcholine plus GTP;(2) direct activation of the GTP-binding proteins with the nonhydrolyzable GTP analog GTP gamma S and direct activation of PKC with the phorbol ester PDBu increased Ca2+ sensitivity in a concentration-dependent manner; and (3) halothane had no effect on the increase in Ca2+ sensitivity induced by GTP gamma S or PDBu.
Regulation of Ca sup 2+ Sensitivity
In many types of smooth muscle, membrane receptor stimulation enhances Ca2+ sensitivity. [2,5,12] In this CTSM preparation, we used acetylcholine plus GTP to submaximally activate m3muscarinic receptor subtypes mediating the contractile response.  Permeabilized smooth muscle preparations have been used as a tool to investigate the mechanisms regulating Ca2+ sensitivity. [8,9,12,30] Compounds to create these preparations include staphylococcus alpha-toxin and the saponin ester beta-escin, [9,13,18,31] which produce pores in the plasma membrane. The advantage of these preparations is that [Ca2+]imay be manipulated by changing the composition of the extracellular solution bathing the smooth muscle. In the present study, [Ca2+]iwas clamped by solutions buffered with EGTA, and as a further precaution, all solutions contained the calcium ionophore A23187, which depletes intracellular Ca2+ stores. As verified in permeabilized vascular smooth muscle by electron probe X-ray microanalysis,  we previously demonstrated using fura-2 fluorescence measurements in this CTSM preparation that these experimental conditions eliminate intracellular Ca2+ gradients and maintain [Ca2+]iconstant during muscarinic receptor stimulation.  Large cellular proteins necessary for contraction, such as calmodulin, myosin light chain kinase, regulatory myosin light chain, actin, and myosin are preserved, and coupling of membrane receptors to second messenger systems that enhance Ca2+ sensitivity is retained and can be activated. [5,8,9,12,18,22,30] For example, in preliminary studies we showed that adding exogenous calmodulin did not affect the contractile response in our CTSM preparation (unpublished observation).
Intracellular second messenger systems are thought to regulate agonist-induced increases in Ca2+ sensitivity, [9,12,13,23] including those involving GTP-binding proteins [10–17] and PKC. [19–22] In our CTSM preparation, increases in Ca2+ sensitivity induced by muscarinic receptor stimulation require GTP, providing evidence for the involvement of GTP-binding proteins in agonist-induced Ca2+ sensitization (data not shown). Furthermore, GTP gamma S, a nonhydrolyzable analog of GTP, mimics [8,11,12,15,17] and GDP beta S, which competitively prevents the binding of guanine nucleotides to GTP-binding proteins, inhibits  agonist-induced Ca2+ sensitization in various smooth muscle types. We confirmed these findings in the present study, showing that GTP gamma S produced a concentration-dependent increase in Ca2+ sensitivity (Figure 3(A)). In the permeabilized CTSM preparation used, GDP beta S markedly reversed the acetylcholine-induced contraction at constant submaximal [Ca2+] sub i (Figure 4). Pre-incubation with GDP beta S also inhibited force development induced by acetylcholine plus GTP. In addition, after extensive permeabilization with Triton X-100, the Ca2+-sensitizing effect of receptor agonists, GTP and GTP gamma S is abolished [10,30](unpublished observation, data not shown).
Two different classes of GTP-binding proteins, heterotrimeric and small, monomeric GTP-binding proteins, are involved in receptor-mediated Ca2+ sensitization. Both the heterotrimeric [10,16] and small molecular weight cytosolic GTP-binding proteins, such as rhoA p21 [10,11,32] or ras p21,  are activated by GTP gamma S. Heterotrimeric GTP-binding proteins induce Ca2+ sensitization in permeabilized smooth muscle preparations. [10,16] Activated ras p21 GTP-binding proteins mimic the Ca2+-sensitizing effect of GTP gamma S and GTP in guinea pig mesenteric microarteries  and adenosine diphosphate ribosylation of the rho family proteins completely abolishes GTP gamma S and agonist-induced Ca2+ sensitization in permeabilized porcine aortic smooth muscle cells  and guinea pig vas deferens.  The current hypothesis suggests that a cascade of GTP-binding proteins may mediate agonist-induced Ca2+ sensitization, with membrane-bound receptors activating heterotrimeric GTP-binding proteins, which in turn activate small cytosolic GTP-binding proteins that affect downstream effector proteins such as protein phosphatases.
It has been suggested that Ca2+ sensitizing agonists acting via GTP-binding proteins increase Ca2+ sensitivity in permeabilized smooth muscle preparations in part by activating PKC. [13,20,21,33] After receptor binding, phospholipase C is activated by a GTP-dependent process, producing diacylglycerol, a physiologic activator of PKC, from phoshatidylinositol biphosphate and phosphatidylcholine.  Once activated, PKC may directly or indirectly inhibit the regulatory myosin light chain phosphatases,  increasing regulatory myosin light chain phosphorylation and force at a constant [Ca2+]i.  This hypothesis is supported by the observation that phorbol esters such as PDBu that activate PKC  increase Ca2+ sensitivity in permeabilized smooth muscle preparations. [16,19,21] We confirmed that PDBu increases Ca2+ sensitivity in a concentration-dependent manner. In preliminary studies (data not shown), we found that contractions induced by PDBu developed only in the presence of [Ca2+]iof 0.1 micro Meter or greater, suggesting that the majority of PKC isoforms involved in agonist-induced Ca2+ sensitization are calcium dependent. Further evidence for the involvement of PKC in agonist-induced Ca2+ sensitization has been provided in other smooth muscle types using PKC antagonists to reverse [31,37] or inhibit  increases in Ca2+ sensitivity.
Consistent with our previous findings, muscarinic receptor stimulation increases Ca2+ sensitivity in CTSM (Figure 1), and halothane significantly inhibits this agonist-induced potentiation in force at constant [Ca2+]i. [2,5] Previously we showed that halothane does not affect free Ca2+ concentration-response curves in the absence of muscarinic receptor stimulation.  During muscarinic receptor stimulation, halothane decreases Ca2+ sensitivity in CTSM in a concentration-dependent manner.  In the current study, halothane had no effect on increases in Ca2+ sensitivity induced by direct activation of GTP-binding proteins with GTP gamma S. This finding can be interpreted in the following ways:
First, because GTP gamma S activates both heterotrimeric and small, cytosolic GTP-binding proteins directly, it might not adequately mimic activation of the particular GTP-binding proteins involved in acetylcholine-induced Ca2+ sensitization in airway smooth muscle. For example, GTP gamma S may activate parallel sensitization pathways not stimulated by acetylcholine.
Second, if the final GTP-binding protein of the signal transduction cascade is activated by GTP gamma S, any halothane effects on GTP-binding proteins upstream of this target GTP-binding protein may not be detected. For example, halothane would not be expected to affect the response to GTP gamma S if it acted to disrupt the coupling between heterotrimeric and small, cytosolic GTP-binding proteins.
Third, halothane could affect processes involving GTP hydrolysis during muscarinic receptor stimulation. Because we used GTP gamma S, the nonhydrolyzable form of GTP, the results of any such effects would not be observed in our experiments. Therefore, we performed additional experiments investigating the effect of halothane on GTP-induced Ca2+ sensitization. Halothane (0.89 +/- 0.12 mM) had no effect at any GTP concentration studied (0.01–1 mM; experiments performed at a constant [Ca2+]iof 0.3 micro Meter; n = 3), which is similar to results obtained using GTP gamma S. This finding suggests that halothane's effect on Ca2+ sensitivity does not depend on GTP hydrolysis.
Finally, when combined with the findings that acetylcholine-induced Ca2+ sensitization can be attenuated by GDP beta S and depends on GTP, the lack of halothane's effect on GTP gamma S-induced Ca2+ sensitization implies that the site of its action may be the coupling between the muscarinic receptor and GTP-binding proteins. Other studies have also suggested that volatile anesthetics may interfere with GTP-binding protein function. [38–40] Further investigations of this mechanism will require better characterization of GTP-dependent pathways regulating Ca2+ sensitivity.
In the present study, halothane did not affect PDBu-induced Ca sup 2+ sensitization. Alterations in PKC activity have been suggested to be a mechanism for general anesthetic effects [3,41–43] in several cell types. Yamakage  found that halothane inhibits the enzyme translocation from the cytosol to the plasma membrane during muscarinic receptor stimulation. Because the membrane fraction is thought to be the active form of PKC, Yamakage suggested that inhibition of PKC activity might be responsible for the halothane-induced decrease in Ca2+ sensitivity. However, because halothane also decreased [Ca2+]iand because many PKC isoforms are Ca2+ dependent, halothane's effect on PKC translocation may simply reflect its effect on [Ca2+]i. We conclude that halothane attenuates Ca2+ sensitivity in permeabilized CTSM during muscarinic receptor stimulation by acting at a site of the signal transduction prior to any activation of PKC.
In summary, in beta-escin-permeabilized CTSM strips, acetylcholine plus GTP, GTP gamma S, and PDBu each increased Ca2+ sensitivity at constant submaximal [Ca2+]i. Halothane significantly inhibited acetylcholine-induced Ca2+ sensitization, whereas it had no effect on PDBu- or GTP tau S-induced Ca2+ sensitization. These results suggest that the site of halothane's action may be the coupling between the muscarinic membrane receptor and GTP-binding proteins, implicating a potentially important anesthetic mechanism in airway smooth muscle.
The authors thank Kathy Street and Robert Lorenz for technical assistance and Janet Beckman and Cathy Nelson for preparing the manuscript.