Halothane Attenuation of Calcium Sensitivity in Airway Smooth Muscle : Mechanisms of Action during Muscarinic Receptor Stimulation

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. [5] 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. [5] 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. [24] 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, [5] 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 [25]) 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 [5]) 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 [26] 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. [27]

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. [28] 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 F^mand 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 F^m, EC⁵⁰, or both parameters. Probability values of 0.05 or less were considered significant.

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.

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.

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. [29] 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, [9] 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. [5] 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 [9] 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, [17] 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 [17] 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 [32] and guinea pig vas deferens. [16] 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. [34] Once activated, PKC may directly or indirectly inhibit the regulatory myosin light chain phosphatases, [35] increasing regulatory myosin light chain phosphorylation and force at a constant [Ca²+]ⁱ. [8] This hypothesis is supported by the observation that phorbol esters such as PDBu that activate PKC [36] 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 [31] 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. [5] During muscarinic receptor stimulation, halothane decreases Ca²+ sensitivity in CTSM in a concentration-dependent manner. [5] 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 [3] 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.