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Anesthesiology:
Laboratory Investigations

Effects of Halothane on the Sarcoplasmic Reticulum Ca2+ Stores and Contractile Proteins in Rabbit Pulmonary Arteries

Su, Judy Y. PhD; Tang, Luo‐Jia MS

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Abstract

Background: The authors' purpose of this study was to elucidate the mechanisms of direct effects of halothane on the contractile proteins and Ca2+ release from the sarcoplasmic reticulum Ca2+ stores using isolated skinned strips (sarcolemma permealized with saponin) from rabbit pulmonary arteries.
Methods: The sarcoplasmic reticular Ca2+ stores were examined by immersing the skinned strips sequentially in solutions to load Ca2+ into and release Ca2+ from the sarcoplasmic reticulum using caffeine, inositol 1,4,5‐trisphosphate, or halothane. The contractile proteins were assessed by activating the strips with Ca2+ followed by administration of halothane (with or without protein kinase C inhibitors). Tension, fura‐2 fluorescence activated by Ca2+ release, and phosphorylation of myosin light chains were measured.
Results: Halothane (0.07–3.00%) increased Ca2+ tension, and phosphorylation of myosin light chains in a dose‐dependent manner. Halothane decreased accumulation of Ca2+ in the sarcoplasmic reticulum and enhanced the caffeine‐induced tension transients. In strips pretreated with caffeine or inositol 1,4,5‐trisphosphate, halothane‐induced tension transients were reduced but Ca2+ was not. In strips activated by 1 micro Meter Ca2+, halothane (0.5–3.0%) decreased 20–45% of the activated force at 15 min. Halothane (3%) transiently increased the force (20%) associated with increases in Ca sup 2+ and phosphorylation of myosin light chains. The increased force was abolished and the subsequent relaxation was enhanced by the protein kinase C inhibitor bisindolylmaleimide but not by indolocarbazole Go‐6976.
Conclusions: In skinned pulmonary arterial strips, halothane, at clinical concentrations, inhibits uptake of Ca2+ by and induces release of Ca2+ from intracellular stores possibly shared by caffeine and inositol 1,4,5‐trisphosphate, which are regulated by phosphorylation of myosin light chains. The time‐dependent inhibition of the contractile proteins by halothane may be mediated by Ca2+ ‐independent protein kinase C.
IN whole animals or humans, halothane has been shown to induce vasoconstriction or vasodilation in different vascular beds, [1] in part by its direct effect on vascular smooth muscle (VSM). In isolated intact arterial (endothelium‐denuded) preparations from various vascular beds and animal species, [2–6] halothane has been shown to cause relaxation. In contrast, halothane induces contraction in isolated pulmonary arterial rings, which is correlated with release of Ca2+ from intracellular stores. [7] The mechanism(s) of this halothane‐induced contraction or relaxation of the VSM is not known.
The primary contractile process in VSM is elevation of cytosolic free Ca2+, from either influx of Ca2+ through the sarcolemma or release of Ca2+ from intracellular stores (the sarcoplasmic reticulum [SR]) by various neurohumoral transmitters. This binds calmodulin, resulting in activation of myosin light chain (MLC) kinase and phosphorylation of MLCs (MLC‐p). This increase in MLC‐p activates myosin adenosine triphosphatase (AT‐Pase), resulting in actin‐myosin interactions and force generation. [8] The release of Ca sup 2+ from the SR occurs through channels that contain receptors specific to either ryanodine or inositol 1,4,5‐trisphosphate (IP3). [9] Whether these two receptor types govern separate Ca2+ stores is not clear, nor is it known if halothane affects them equally. In contractile proteins, Ca2+ ‐independent pathways have been demonstrated to be regulated either by MLC‐p [10] or by the thin filament‐associated proteins calponin and caldesmon. [11,12] These multiple regulatory mechanisms in VSM contraction may contribute to the diversity of vascular responses to halothane.
At the intracellular sites of the contractile process, we have shown in saponin‐skinned aortic strips that halothane enhances caffeine‐induced tension transients. [3] This suggests that halothane causes release of Ca2+ from the SR or increases the sensitivity of the contractile proteins to Ca2+. In cultured VSM cells, direct release of Ca2+ from the intracellular Ca2+ stores [13] and inhibition of inositol phosphate formation [14] by halothane have been observed. Therefore, these multiple effects of halothane on intracellular signaling would result in opposing effects. In isolated intact bovine pulmonary arterial rings treated with SR ATPase inhibitor (cyclopiazonic acid) or an SR Ca2+ release channel inhibitor (ryanodine), halothane and caffeine decrease contraction, [7] which suggests halothane‐induced release of Ca2+ from the caffeine‐releasable SR Ca2+. Whether halothane affects VSM contraction by releasing Ca2+, either directly or indirectly, from IP3‐releasable stores is not clear.
At the contractile protein level, halothane has been shown to decrease submaximum and maximum Ca2+ ‐activated force of the contractile proteins in sarcolemma‐permealized rat mesenteric arterial strips. [5] Recently, the inhibition of protein kinase C (PKC) activator‐induced contraction by halothane has been observed in isolated rat coronary arteries, [15] which could result in decreased influx of Ca2+ via the sarcolemma [16] or depress force development by a Ca2+ ‐independent pathway. [10–12] The mechanisms of the Ca2+ ‐independent effect of halothane are not clear, however.
Accordingly, in this study, we examined intracellular mechanisms (either SR or the contractile proteins) of action of halothane using saponin‐skinned (sarcolemma‐permealized) arterial strips [3] from rabbit pulmonary artery. This skinned strip preparation allowed us to examine the function of each organelle and to measure tension, free Ca2+ concentrations, and MLC isoforms simultaneously.
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Materials and Methods

Skinned Arterial Strips
New Zealand male White rabbits (2.0–2.5 kg) were killed using a captive bolt pistol (approved by the Institutional Animal Care Committee of the University of Washington, Seattle, WA), followed by exsanguination. Right or left pulmonary arteries were rapidly and carefully isolated and kept on ice until experimentation.
Skinned strips were prepared in a manner reported previously for aorta. [3] The strips (0.1–0.15 mm wide) were mounted on photodiode transducers, stretched to a 50‐mg tension from the resting length and then immersed in a skinning solution (a relaxing solution containing 0.3 mg/ml saponin) for 5 min. Isometric tension of the skinned strips was recorded on a computer (Quadra 950; Apple Computer, Inc., Cupertino, CA) with a customized LabVIEW software program interfaced with a multifunction I/O board with 16‐bit resolution (NB‐MIO‐16XL; National Instrument, Austin, TX). The experiments were performed at room temperature (20 [degree sign] Celsius‐23 [degree sign] Celsius).
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Study of Uptake or Release of Ca sup 2+ from the Sarcoplasmic Reticulum
Figure 1
Figure 1
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The skinned strips were immersed sequentially in four solutions to load Ca2+ into and to release Ca2+ from the SR (a load‐release cycle; Figure 1). These four solutions contained the same ionic concentration (35 mM K sup +, 35 mM Na sup +, 15 mM creatine phosphate, 2 mM MgATP2‐, 0.1 mM Mg2+, 80 mM methanesulfonate, and 50 mM piperazine‐N,N'‐bis[2‐ethanesulfonic acid dipotassium; ionic strength, 0.15; pH 7.00), except that concentrations of EGTA and Ca2+ varied as follows. Solution 1 (no added Ca2+, 7 mM EGTA) was used to wash away saponin in the strips or caffeine in subsequent load‐release cycles; solution 2 (0.316 micro Meter Ca2+, 7 mM EGTA) was used to load Ca2+ rapidly into the SR; solution 3 (0.1 micro Meter Ca2+, 0.05 mM EGTA) was used to reduce EGTA in the strips; and solution 4 (same as solution 3 plus 10 mM caffeine for controls and various concentrations of halothane +/‐ caffeine for tests) was used to induce release of Ca2+ from the SR as measured with a tension transient or Ca2+ fura‐2 fluorescence (described later). The duration of immersion was 5 min for solutions 1 or 2 and 15 min for solution 3, and the strips remained in solution 4 until the generated tension transient had returned to its steady‐state baseline value (Figure 1).
Figure 2
Figure 2
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Halothane was present in two different aspects of the study. In the first study, halothane was used either to modulate the magnitude of caffeine‐induced release of Ca2+ (Figure 1) or to stimulate release of Ca2+ from the SR directly in the absence of caffeine. In the second study, the direct Ca2+ release effect of halothane was examined again in the absence of caffeine, but the Ca2+ stores in the SR were manipulated by prior treatment with caffeine or IP3 (Figure 2).
Effect of Halothane on Uptake or Release of Ca2+ from the Sarcoplasmic Reticulum. Three load‐release cycles were performed in each skinned strip: a control cycle (Ca2+ releasing solution 4 contained 10 mM caffeine), a test cycle (solutions contained halothane), and finally another control cycle. The test cycle consisted of one of the following conditions: (1) the uptake phase, in which halothane was present during Ca2+ loading into the SR in solutions 1–3; or (2) the release phase, in which halothane was present during release of Ca sup 2+ from the SR in solution 4 with or without caffeine. The area of tension transient in the test cycle was expressed as a percent of that of the mean of the bracketing controls in each preparation.
A halothane and N2 mixture was delivered through a Verni‐Trol vaporizer (Ohio Medical Product, Madison, WI) for test solutions, and control solutions were saturated with 100% N2. The halothane concentrations in the final solutions were assayed by gas chromatography and expressed in partial pressure as a percent of one atmosphere. [17] The calculated concentration of halothane in the gas was 0.41 mM for 1% vapor concentration, or 7.6 mmHg partial pressure of one atmosphere (760 mmHg at sea level) based on n/V = P/RT, where n/V is the molar concentration of halothane, R (gas constant) is 0.082, T ([degree sign] K) (absolute temperature) is 296 [degree sign] K at 23 [degree sign] Celsius, and P is partial pressure of halothane expressed as a fraction of atmospheric pressure. Assuming a solution/gas ratio of 1.5 at 23 [degree sign] Celsius, the halothane concentration in the solution was 0.6 mM.
Effect of Halothane on Release of Ca2+ from Inositol 1,4,5‐Trisphosphate‐ or Caffeine‐Releasable Stores. In this study, we examined whether halothane releases Ca2+ from specific intracellular stores: the IP3 ‐or caffeine‐releasable store.
The experimental protocol consisted of two load‐release cycles in the following sequence: (1) a control cycle (solution 4 contained 10 mM caffeine), and (2) a test cycle to deplete the Ca2+ stores (solution 4 with 25 mM caffeine or 30 micro Meter IP3 for 15 min followed by three washes in solution 3 of 5 min each), followed by 0.3%, 1.0%, or 3.0% halothane (in solution 3) to release residual Ca2+ (Figure 2). The test results were expressed as a percent of that of 10 mM caffeine in the control cycle.
Measurement of Fura‐2 Fluorescence. Fura‐2, a synthetic fluorescent dye, [18] was selected to measure free Ca2+ concentration during muscle contraction because of several advantages including (1) sensitivity to [Ca2+] in the physiologic range, i.e., > 0.01 micro Meter; (2) selectivity for Ca2+; (3) the low concentration of fura‐2 required (micro Meter range) reduces the possibility of calcium buffering by the indicator; and (4) the ratio of fluorescence intensities from 340 (F340) to 380 nm (F380) excitation wavelength for [Ca2+] calculation eliminates variabilities of instrument efficiency, dye concentration, and effective cell thickness along the optical beam.
The validity of the measurement of fura‐2 initially was confirmed by titrating 10 mM Ca2+ EGTA into 10 mM EGTA buffer (Calcium Calibration Buffer Kit 1, C‐3008; Molecular Probes, Inc., Eugene, OR). A dissociation constant for fura‐2/Ca2+ complex (Kd) of 173 nM was obtained in our laboratory using a modified calcium analyzer (CAF‐100; Japan Spectroscopic, Tokyo, Japan), which is of a similar magnitude (145 nM) to that reported by Molecular Probes. Next, the calibration of Ca2+ was performed in the presence of the skinned strip during our experimental condition by equilibrating with 0.05 mM EGTA buffer (solution 3 in the load‐release cycle) containing various concentrations of Ca2+ plus 2 micro Meter fura‐2. Based on the equation of Ca2+ (nM) = Kd x (Sf2 /Sb2) x [(Ri ‐ Rmin)/[(Rmax ‐ Ri)], [18] the apparent Kd value was calculated from the best linear fit with log (Sf2 /Sb2) x [(R sub i ‐ Rmin)/(Rmax ‐ Ri)] as the y axis, and log [Ca2+] (M) as the x axis. Sf2 is the fluorescence emitted at F380 at 0 Ca2+ during which fura‐2 was in free form, Sb2 is the F sub 380 at 0.1 mM Ca2+ during which fura‐2 was in Ca2+ bound form, Ri is the F340‐to‐F380 ratio at various concentrations of free Ca2+, Rmin is the F340‐to‐F380 ratio at 0 Ca2+, and Rmax is the F340‐to‐F380 ratio at 0.1 mM Ca2+. The calibrated data were (mean +/‐ SEM [n = 5]) Kd, 252.2 +/‐ 12.2 nM; Sf2, 1.23 +/‐ 0.1; Sb2, 0.48 +/‐ 0.04; Rmin, 0.06 +/‐ 0.003; and Rmax, 0.56 +/‐ 0.049.
Figure 3
Figure 3
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The experimental protocol was the same as described for tension measurement except that (1) all bathing solutions contained 2 micro Meter fura‐2 free acid, and fura‐2 fluorescence was measured before (baseline) and during release of Ca2+; (2) halothane was prepared in a polyethylene centrifuge tube containing solution 3, and the concentration of halothane in the solution was assayed by gas chromatography. [17] Simultaneous measurements of tension and fura‐2 fluorescence (Figure 3) were performed by placing the skinned strip into a 150‐micro liter quartz tissue bath containing solution 3 as baseline fluorescence (described later). Next, a releasing solution was injected (solution 4 of the control cycle or the test solution of the test cycle).
The fluorescence of fura‐2 (F340 and F380) was measured continuously by exposing the skinned strip alternately, at 50 Hz, to 340 +/‐ 10 and 380 +/‐ 10 nm excitation wavelengths. The fluorescence emitted at the 500 +/‐ 20 nm wavelength was detected using a modified calcium analyzer (CAF‐100; Japan Spectroscopic, Tokyo, Japan). The fluorescence emitted from excitation wavelengths of 340 and 380 nm were recorded, at a sampling rate of 10 Hz, on a PowerMac 7100 (Apple Computer, Inc.) using a customized LabVIEW software program interfaced with a multifunction I/O board with 16‐bit resolution (NB‐MIO‐16XL; National Instrument).
Using these data from Ca2+ calibration in skinned strips and the Ri between baseline (solution 3 of the load‐release cycle in the presence of a skinned strip) and the peak (3 s after administration of halothane; Figure 3), the amount of free Ca2+ released from the SR was estimated from the difference in Ca2+ between the baseline and the peak.
When the skinned strip was immersed in solution 3 (0.05 mM EGTA with no added Ca2+) of the load‐release cycle, the calculated Ca2+ from the ratio of fura‐2 fluorescence in this solution (Figure 3(C)) was [nearly =] 118 nM, which agrees with the [nearly =] 100 nM Ca2+ derived from the Calcium Buffer (Calcium Calibration Buffer Kit I, C3008; Molecular Probes Inc.).
In each skinned strip, the result from the test cycle was expressed as a percent of that of 10 mM caffeine from the control cycle. Analysis of variance was used to compare the test results regarding various concentrations of halothane. A probability value < 0.05 was regarded as statistically significant.
The concentrations of Ca2+ in the buffer for calibration of Ca2+ were made by mixing 0.05 mM EGTA buffers containing 0.1 mM Ca2+ and 0.1 micro Meter (estimated from the Ca2+ standard of Molecular Probes in solution 3 of the load‐release cycle) to obtain the intermediate concentrations of Ca2+.
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Study of Ca sup 2+ Activation of the Contractile Proteins
Figure 4
Figure 4
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Direct effects of halothane on submaximum Ca2+ ‐activated force development (1 micro Meter Ca2+, buffered with high EGTA) of the skinned strips and the degree of MLC‐p were investigated. When the force reached a steady state, the same solution containing a specific concentration of halothane was injected into a tissue bath and the effects of halothane were observed for 15 min (Figure 4). The peak (within 1 min) force development and that at 15 min after administration of halothane were expressed as a percent of the control (before the administration of halothane). Parallel experiments were performed as time controls by administration of solution without halothane.
In different experiments, skinned strips were tested with (test group) or without (time control group) halothane (described previously), and at 1 min or 15 min the strips were quickly frozen in freon (‐130 +/‐ 30 [degree sign] Celsius) cooled with liquid N2 for quantification of MLC isoforms.
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Quantification of Myosin Light Chain Isoforms by Two‐dimensional Electrophoresis and Immunoblotting
The method of extraction and separation of MLC isoforms from VSM was the same as that described by Kitazawa et al. [19] Myosin light chain proteins were extracted from the frozen strips. This was followed by separation of MLC isoforms by two‐dimensional polyacrylamide gel electrophoresis for its high resolution and sensitivity, [20] by their charge using isoelectric focusing polyacrylamide gel electrophoresis, and then by their mass (molecular weight, 20 kDa) using sodium dodecyl sulfate polyacrylamide gel electrophoresis in the second dimension.
Immunoblotting was performed as described by Hathaway and Haeberle [21] by transferring sodium dodecyl sulfate gel onto nitrocellulose membranes by transblot electrophoresis, and MLC isoforms were specifically labeled with polyclonal affinity‐purified rabbit anti‐MLC antibody (supplied by Dr. Susan Gunst of the Department of Physiology, Indiana University, Indianapolis, IN). This was followed by exposure to a second antibody labeled with horseradish peroxidase (antirabbit immunoglobulin G peroxidase conjugate; Sigma Chemical Co., St. Louis, MO) to react with the immobilized protein antigen (MLC), which was then detected on nitrocellulose membranes by autoradiography.
Determination of the extent of MLC‐p in bands (immunoblots) was done with a multiresolution scanning imaging densitometer and the Molecular Analyst software program (model GS‐700; Bio‐Rad Laboratories, Hercules, CA).
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Data Analysis
Areas of tension transients from load‐release cycles or submaximum Ca2+ ‐activated force at peak or steady state were calculated. The test results were expressed as a percent of those of the control results for both the test and time control experiments. The amount of MLC‐p was expressed as a percent of the total MLC.
The test results were compared with those of controls using Student's t test for unpaired data. Using the Stat‐VIEW software program (BrainPower, Inc., Calabasas, CA), Student's t test for unpaired data was used to compare the results from the test experiments and the time controls within each concentration of halothane, and two‐factorial analysis of variance was used to compare concentrations of halothane. Data are expressed as mean +/‐ SEM (n). A probability value < 0.05 was considered significant. [22]
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Materials
Fura‐2 pentapotassium salt and IP3 were purchased from Molecular Probes. Thymol‐free halothane was supplied by Halocarbon Laboratories (Hackensack, NJ). Antiserum for MLC was supplied by Dr. Susan Gunst (University of Indiana, Indianapolis, IN). Protein kinase C inhibitors (Go‐6976 and bisindolylmaleimide I‐HCl) were purchased from Calbiochem (La Jolla, CA). Go‐6976 was made in 100% dimethylsulfoxide. Caffeine was purchased from Sigma Chemical Co., and other chemicals were analytical or reagent grade.
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Results

Effects of Halothane on Uptake or Release of Ca sup 2+ from the SR Measured with Caffeine‐induced Tension Transients
Figure 5
Figure 5
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We found that halothane (0.5%, 1.0%, and 2.0%) in the uptake phase (experimental protocol shown in Figure 1) decreased the caffeine‐induced tension transients by 10–80% in a dose‐dependent fashion (Figure 5). In contrast, when halothane was present in the release phase (experimental protocol shown in Figure 1) the caffeine‐induced tension transients significantly increased 40–100% and reached a maximum at 1% halothane (Figure 5).
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Direct Effect of Halothane on Tension, Ca sup 2+, and Phosphorylation of Myosin Light Chains
In skinned pulmonary arterial strips, 10 mM caffeine produced a peak Ca2+ concentration of 37.7 +/‐ 5.3 nM (n = 24). This amount of Ca2+ release is in agreement with that observed in permealized rabbit mesenteric arterial strips. [23]
Figure 6
Figure 6
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Figure 7
Figure 7
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When halothane (0.1%, 0.3%, 1.0%, and 3.0%, no caffeine) was used to stimulate release of Ca2+, tension transients (experimental protocol shown in Figure 1, a typical tracing shown in Figure 3(D)) were produced in a dose‐dependent manner (Figure 6). The tension transients were correlated with increases in release of Ca2+ from the SR (tracings shown in Figure 3(A, B, and C); average results shown in Figure 6). The Ca2+ transients, however, appear to increase proportionally more at the lower concentrations of halothane and proportionally less at 3% halothane. This halothane‐induced tension transient and release of Ca2+ were associated with increased MLC‐p (30% of total MLC after 1 min of 1% halothane; Figure 7(B)) compared with the time control group, which showed no phosphorylation at 0% halothane (Figure 7(B)). After 7 min of 1% halothane, tension and myosin MLC‐p had returned to baseline (Figure 7).
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Differentiation of Possible Ca sup 2+ Stores in the Sarcoplasmic Reticulum Released by Halothane and its Mechanisms of Action
Figure 8
Figure 8
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This study was performed to determine further whether the halothane‐induced release of Ca2+ from the SR was from a specific SR store (caffeine‐ or IP3 ‐releasable store). In strips pretreated with either caffeine or IP3 without reloading of Ca2+ into the SR (experimental protocol shown in Figure 2), the halothane‐induced tension transients were reduced greatly (averages of 80–90%, 90%, and 50–70% for 0.3%, 1.0%, and 3.0% halothane, respectively, Figure 8(A)) compared with those in strips without pretreatment (Figure 8(A)). The reduction was similar between caffeine and IP3‐treated strips (Figure 8(A)). In contrast, the degree of Ca2+ released by halothane was not significantly changed during either condition nor in all three concentrations of halothane tested (Figure 8(B)).
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Effects of Halothane on Submaximum Ca sup 2+ Activation of the Contractile Proteins
Figure 9
Figure 9
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At 1 micro Meter Ca2+, skinned pulmonary arterial strips generated a steady‐state force of 35.2% +/‐ 2.4 (n = 10) of the maximum tension. The initial effect of halothane was no change in force except at 3% halothane, at which a transient increase (20%) in force was observed (tracings shown in Figure 4; average results shown in Figure 9). Within 15 min of halothane exposure, the submaximum Ca2+ ‐activated force was decreased in a dose dependent manner (average of 22–44%, Figure 9(A)).
The initial increase in force produced by 3% halothane was accompanied by increases in concentrations of Ca2+ (data not shown) and MLC‐p (Figure 9(B)). In contrast, the halothane‐induced decrease in force at 15 min was not associated with changes in MLC‐p (Figure 9(B)).
Figure 10
Figure 10
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The role of PKC in this halothane‐induced relaxation of the submaximum Ca2+ ‐activated force was investigated further using inhibitors of PKC. We found that Go‐6976, a specific inhibitor of the Ca sup 2+ ‐dependent PKC isozymes alpha and beta I, [24] did not affect the initial increase or subsequent decrease in force by 3% halothane (Figure 10). The initial increase was blocked, however, and the subsequent decrease in force was enhanced by bisindolylmaleimide I‐HCl (Figure 10), an inhibitor of the alpha, Beta I, beta II, gamma, and epsilon isozymes of PKC. [25]
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Discussion

This study shows that halothane has complex effects in skinned rabbit pulmonary arterial strips. The major findings are that (1) halothane activates release of Ca2+ from the intracellular stores, although prerelease of intracellular stores with caffeine and IP sub 3 has a modest effect on this process; (2) all concentrations of halothane appear ultimately to cause a time‐dependent depression of tension development that is not associated with decreased MLC‐p; and (3) halothane at high concentrations induces a transient activation that may involve more than simple release of Ca2+.
The direct halothane‐induced tension transient is associated with increased release of Ca2+ from the SR. The increased cytosolic Ca2+ binds to calmodulin and activates Ca2+ /calmodulin‐dependent MLC kinase. This activated MLC kinase phosphorylates the MLCs resulting in increased actomyosin ATPase activity, actin‐myosin interaction, and finally force generation. [8] The halothane‐induced release of Ca2+ from the SR also is observed in isolated intact bovine pulmonary artery. [7] The direct release of Ca2+ from the SR by halothane contributes, at least in part, to the enhancement of caffeine‐induced tension transients when halothane is present in the release phase. Direct release of Ca2+ from the SR also could result in less accumulation of Ca2+ in the SR when halothane is present in the Ca2+ uptake phase and thus might explain the decreased caffeine‐induced tension transients. It is also possible, however, that halothane directly inhibits SR ATPase as demonstrated in striated muscle, [26] but this remains to be confirmed in VSM.
Based on the dose‐response relation of halothane (Figure 6) on tension and Ca2+ (normalized by the 10 mM caffeine data), the higher percent increase in release of Ca2+ than that of tension induced by 0.1% and 0.3% halothane suggests that there is a direct Ca sup 2+ independent halothane‐induced relaxation. The direct halothane‐induced relaxation observed during the low EGTA condition is further substantiated by halothane‐decreased submaximum Ca2+ ‐activated force in the Ca2+ clamped condition (7 mm EGTA). In contrast, a greater increase in force than Ca2+ by 1% and 3% halothane (Figure 6) suggests that halothane increases the sensitivity of the contractile proteins to Ca2+, which agrees with our observations that high concentrations of halothane (> 5%) induced contracture with little change in Ca2+ in skinned strips treated with Ca2+ ionophore (A23187; data not shown). These speculations, however, remain to be proved.
In the study of Ca2+ stores pretreated with either 25 mM caffeine or IP3 without reloading, the significant decrease in tension without a statistically significant change in Ca2+ caused by 1% and 3% halothane may be due to the insensitivity of fura‐2 measurement to detect the small decrease in Ca2+. This speculation is based on the dose‐response relation of halothane in untreated strips (Figure 6), in which a small (not statistically significant) decrease in Ca2+ is accompanied by a large decrease in tension between 1.0% and 0.3% halothane. It is possible that in treated strips this curve is shifted to the right of that for untreated strips, so that the steepest part of the curve is now between 1% and 3% halothane. Moreover, there is a trend toward a decrease in release of Ca2+ by 1% and 3% halothane in caffeine‐ and IP3 ‐treated strips compared with untreated strips. If this speculation is correct, the markedly decreased tension in caffeine‐ and IP3 ‐treated strips could be the result of combined decreases in sensitivity of the contractile proteins and small decreases in release of Ca2+ by halothane. Nonetheless, the finding that there is no difference between caffeine‐ and IP3 ‐treated strips suggests that halothane releases Ca2+ from both caffeine‐ and IP3 ‐releasable stores.
The lack of change of halothane‐induced Ca2+ transients in contrast to decreases in tension transients in strips treated with caffeine or IP3 is not likely due to halothane‐induced release of Ca2+ from mitochondria, because strips treated with sodium azide (an inhibitor of the mitochondria pump) do not show reduced halothane‐ or caffeine‐induced tension transients but rather increased tension transients (data not shown). This phenomenon also has been shown in skinned myocardial fibers. [27] These observations suggest that, in our experimental conditions, mitochondria may play a role in the reuptake of Ca2+ released by halothane or caffeine but not in the release of Ca2+. A localization of release of Ca2+ by halothane may reveal whether halothane releases Ca2+ from stores that are not releasable by caffeine and IP3.
This lack of significant change of halothane‐induced release of Ca2+ in caffeine or IP3 ‐treated strips, however, is not due to the lack of validity of the fura‐2 fluorescence measurement as evidenced by the following. First, the amount of Ca2+ release by 10 mM caffeine is in the same magnitude reported in beta‐escin‐treated rabbit mesenteric arteries. [23] Second, halothane releases Ca2+ in a dose‐dependent fashion, accompanied with tension transients (Figure 6). Third, in the same conditions, Ca2+ and tension transients are abolished in strips treated with Triton‐X‐100 (Sigma, St. Louis, MO) (data not shown). Therefore, in our experimental conditions, it is reasonable to assume that tension transients reflect the amount of Ca2+ release from the Ca2+ stores in the SR (Figure 6). Using this assumption, the similar degree of reduction of halothane‐induced tension transients in caffeine‐ or IP3‐pretreated strips, because of either small reductions of Ca2+ transients (not statistically significant, Figure 8(B)) or direct inhibition of the contractile proteins by halothane (discussed later), suggests that the SR stores are shared by ryanodine and IP3 receptor channels. Thus, this release of Ca2+ from the SR by halothane from both caffeine‐ and IP3 ‐releasable Ca2+ stores could be mediated either by activation of the ryanodine receptor SR Ca2+ release channel as observed in isolated cardiac SR [28] or by increases in SR membrane permeability to Ca2+. The resulting increased cytosolic free Ca2+ also would enhance ryanodine depression of caffeine‐induced tension transients as observed in striated muscle. [3,29] This remains to be confirmed in VSM, however.
The 3% halothane‐induced transient increase in sub‐maximum Ca sup 2+ ‐activated force (Figure 4) associated with increases in fura‐2 fluorescence and MLC‐p suggests that a large amount of Ca2+ (mainly buffered by 7 mM EGTA) is released by 3% halothane. Based on the [Ca2+]‐tension relationship (data not shown), the increased fura‐2 fluorescence by 3% halothane is not large enough to account for the force development. This increase in force is greater than the increase in Ca2+, a result consistent with the direct halothane‐induced activation of the contractile proteins suggested in the dose‐response relation of halothane.
The halothane‐induced depression of the submaximum force under the Ca2+ clamped condition without decreases in MLC‐p suggests that mechanisms other than a decreased MLC kinase to MLC phosphatase activity ratio underlie its action. This halothane‐induced relaxation in submaximum Ca2+ ‐activated force can not be due to leak of soluble calmodulin from our preparations, because exogenous administration of calmodulin (up to to 0.1 mM) did not affect either the 1 micro Meter Ca2+ ‐activated force or halothane‐induced relaxation (data not shown). Halothane‐induced relaxation accompanied with no change in MLC‐p also could be due to a hyperbolic relationship between MLC‐p and isometric force. It is possible that the force (35% of the maximum) generated by 1 micro Meter Ca2+ is at the steepest part of the curve so that a small decrease in MLC‐p (not measurable by two‐dimensional electrophoresis) results in significant decrease in force. It is also possible, however, that the force generated during low concentration of Ca2+ is, in part, contributed indirectly by a Ca sup 2+ ‐dependent pathway (such as calmodulin‐dependent protein kinase II) other than MLC‐p. [30]
Because halothane‐induced relaxation occurs 15 min after administration of halothane when the concentration of halothane was either maintained or greatly decreased, it is reasonable to speculate that halothane triggers a cascade pathway resulting in inhibition of the contractile proteins. This cascade pathway could be triggered by a small release of Ca2+ by halothane or could be independent of Ca2+. The blockade of initial increased force by 3% halothane and enhancement of halothane‐induced delayed relaxation by the inhibitor of both Ca2+ ‐dependent and Ca2+ ‐independent isozymes (alpha, beta I, beta II, gamma, and epsilon) of PKC but not by the inhibitor of specific Ca sup 2+ ‐dependent PKC‐alpha or PKC‐beta I suggests that inhibition of one or more of the Ca2+ ‐independent isozymes, possibly epsilon isozyme, may account for halothane‐induced relaxation. The identification of PKC‐alpha and PKC‐epsilon, in collaboration with Liao et al., [31] (data not shown) in our preparation substantiates this speculation. Moreover, an inhibition of PKC by halothane also has been suggested in isolated intact rat coronary arterial preparations contracted by a PKC activator. [15] Whether halothane directly inhibits PKC‐epsilon and whether it is regulated by calponin [32] remain to be examined. It is also possible that halothane‐induced relaxation is caused by decreasing the “latch state” or cross‐bridges cycling rate (for review see [12]), and thus, the interaction between actin and myosin.
Although halothane has multiple actions at the intracellular sites of VSM, during normal conditions, halothane‐induced release of Ca sup 2+, and thus contraction, would play a major role in pulmonary arteries. [7] The halothane‐induced relaxation, however, could be manifested during abnormal or diseased conditions, such as hypoxic pulmonary vasoconstriction [33] with depleted Ca2+ in the SR or abnormal activation of PKC.
In summary, we have shown in rabbit skinned pulmonary arterial strips that halothane (0.1–3.0%) induces release of Ca2+ from intracellular stores associated with increased MLC‐p and tension generation. At clinical concentrations (< 3%), halothane releases Ca2+ from stores shared by both caffeine and IP3. During the submaximum Ca2+ activated state, halothane causes relaxation of the skinned strips by inhibition of Ca2+ ‐independent PKC.
The authors thank Dr. Alec Rooke for discussion and Barbara Pearson for editorial assistance with the manuscript. The supply of thymol‐free halothane by Peter Haines (Halocarbon Laboratories, Hackensack, NJ), supply of myosin light chain antibody by Dr. Susan Gunst (University of Indiana, Indianapolis, IN), supply of calmodulin by Dr. Dean Malencik (Oregon State University, Corvallis, OR), and identification of protein kinase C in skinned arterial strips by Dr. Duan‐Fang Liao (Dr. Bradford Berk's laboratory, University of Washington, Seattle, WA) are greatly appreciated.
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Keywords:
Caffeine; fura‐2 fluorescence; inositol 1,4,5‐trisphosphate; myosin light chains; protein kinase C

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