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MUSCLE CELL BIOLOGY AND CELL MOTILITY
1Department of Pharmacology, Southern Illinois University School of Medicine, Springfield, Illinois; 2Department of Physiology, Loyola University Chicago, Maywood, Illinois
Submitted 19 December 2007 ; accepted in final form 25 February 2008
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ABSTRACT |
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volatile anesthetics; dantrolene; sarcoplasmic reticulum; excitation-contraction coupling; malignant hyperthermia
Several hundred mutations in RyR1 have been mapped and characterized. They were shown to cosegregate with phenotypes corresponding to several diseases, including malignant hyperthermia (MH) susceptibility, central core disease, and multi-minicore disease (reviewed in Refs. 2 and 38).
Although the primary target of the volatile anesthetic halothane is the central nervous system, it has been found that this anesthetic triggers MH episodes. It is apparent that, in MH-susceptible individuals, RyR1-mediated calcium release from skeletal SR is hypersensitive to halothane (reviewed in Refs. 2, 38, and 51). Indeed, [3H]ryanodine binding experiments (37) as well as channel recordings in planar bilayers (28) confirmed RyR1 abnormal behavior and halothane-induced activation of mutated but not wild-type RyR1. Consequently, halothane was chosen as an MH susceptibility test agent for both the North American caffeine halothane contracture test and the European in vitro contracture test (1, 30, 44). On average, dissected muscle fiber bundles taken from MH-susceptible individuals develop contracture in response to low doses of halothane-caffeine. However, there is variability and the responses of MH-susceptible fibers have a degree of overlapping with those of normal fibers. Thus a limitation of these tests is misdiagnosis due to their relatively low specificity (1, 23, 44).
Previous findings suggest that some factors that could change with altered metabolism, such as cytosolic Mg2+ levels and lumenal Ca2+ content, could make wild-type muscle fibers responsive to halothane (11). Indeed, halothane has been shown to largely stimulate RyR1-mediated Ca2+ release from SR vesicles isolated from wild-type rabbit skeletal muscle (33). It is important to mention that while these Ca2+ release studies were performed with high lumenal Ca2+ and in the presence of cytosolic modulators (Mg2+-ATP) (33), the bilayer experiments reporting lack of halothane modulation of wild-type RyR1 were done using simple (Cs+ or K+) solutions (28). It has been reported that the nature of the current carrier as well as the presence or absence of Mg2+-ATP may affect the RyR1 behavior (7, 10, 14). Thus we hypothesized that halothane modulation of RyR1 is highly sensitive to these environmental factors and that the discrepancies between reports on the action of halothane on wild-type RyR1 (28, 33) may reflect differences in the channel environment. Therefore, we investigated how changes in cytosolic levels of ATP and Mg2+, as well as lumenal Ca2+, affect the action of halothane on wild-type RyR1 reconstituted in planar bilayers. We compared the effects of halothane on skeletal RyR1 to those on cardiac RyR2 since, unlike RyR1, RyR2 was reported to be responsive to halothane when isolated in simple bilayer solutions (5) as well as in myocardial cells (17, 45). Since the muscle relaxant dantrolene is effective for the treatment of MH (21, 47) and it may target RyR1 (15, 27, 43), we also tested whether this agent interferes with the action of halothane on RyR channels. We found that halothane affects the behavior of both RyR isoforms. Still, the action of halothane on skeletal RyR1 depends on the levels of physiological cytosolic modulators (Ca2+, Mg2+, and ATP) as well as on lumenal Ca2+ content. Some of the results have been presented in preliminary form (9).
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METHODS |
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30 µM in HEPES-Tris. Halothane stock solutions were prepared fresh at the beginning of each experiment by vigorously mixing halothane and HEPES-Tris (1:3 vol/vol) for 20–60 min. The mixture is allowed to rest on ice until the halothane-saturated HEPES-Tris phase (20 mM halothane, estimated using gas chromatography at Carbon Dynamics) separates from the halothane phase. Halothane stock solutions were kept on ice in vials sealed with a silicon-Teflon septum cap. Sarcoplasmic reticulum vesicle preparation. All procedures with animals were designed to minimize pain and suffering and conformed to the guidelines of the National Institutes of Health. The laboratory animal care and use committee (LACUC) of Southern Illinois University School of Medicine reviewed and approved these protocols for animal use.
Skeletal and cardiac SR microsomes (six different preparations each) were used as a source of RyR1 and RyR2, respectively. Microsomes rich in RyR1 channels (terminal cisternae fraction, TC microsomes) were isolated from predominantly fast-twitch skeletal muscle (back and leg; adult New Zealand White rabbits), as previously described (39). Heavy SR membrane fractions from a dog ventricle, rich in RyR2 channels, isolated using cellular subfractionation methods based on Chamberlain et al. (3), were kindly provided by Dr. S. Fleischer (Dept. Biological Sciences, Vanderbilt University). All preparations were kept in liquid nitrogen. Aliquots (15 µl each) of membranes were prepared every month and stored at –80°C. For experiments, aliquots were quickly defrosted in water, kept on ice, and used within 5 h.
Ryanodine receptor channel recordings and data analysis.
Reconstitution of RyR1 and RyR2 in planar lipid bilayers was done as previously described (6, 7). Briefly, planar lipid bilayers were formed on 80- to 150-µm diameter circular holes in Teflon septa, separating two 1.3-ml compartments. The trans compartment was filled with a HEPES-Ca2+ solution [HEPES 250 mM, Ca(OH)2 53 mM; pH 7.4] and subsequently clamped at 0 mV by an Axopatch 200B patch-clamp amplifier (Axon Instruments, Foster City, CA). The cis compartment (ground) was filled with HEPES-Tris solution (250 mM HEPES, 120 mM Tris, pH 7.4). Bilayers of a 5:4:1 mixture of bovine brain phosphatidylethanolamine, phosphatidylserine, and phosphatidylcholine (45–50 mg/ml in decane) were painted onto the holes of the Teflon septa from the cis side. To promote vesicle fusion, 500–1,000 mM CsCl and 1 mM CaCl2 were added to the cis solution. Cardiac or skeletal SR microsomes (5–15 µg) were then added to the cis solution. After RyR currents (or Cl– currents >100 pA at 0 mV) were observed, the cis chamber was perfused for 5 min at 4 ml/min with HEPES-Tris solution. A mixture of BAPTA and dibromo-BAPTA was used to buffer free [Ca2+] on the cytosolic surface of the channel ([Ca2+]cyt) (6). Free [Mg2+] in mixtures of Mg2+ and ATP was estimated using Winmaxc2.5 by Chris Patton, Stanford University (available for free download at http://www.stanford.edu/cpatton/maxc.html). Unless otherwise stated, "presence of Mg2+/ATP" means total Mg2+ = 4 mM; total ATP = 5 mM (free [Mg2+] = 0.25 mM).
Cardiac and skeletal RyR channels were identified by their unique characteristics of slope conductance (from 80 to 100 pS) and reversal potential (+40 to +50 mV; trans-cis). Channel modulation by agonists, blockers, and conductance modifiers was also assessed as previously described (6). RyR channel currents are depicted as positive (upward deflections) in figures and reflect cation flux from trans (luminal) to cis (cytosolic) compartments.
Recordings of RyR activity (4 to 8 min in duration) were carried out at 0 mV, filtered through a low-pass Bessel filter at 1–10 kHz, digitized at 20–100 kHz with a 12-bit analog to digital converter, and stored on DVD for computer analysis, using the pClamp9 software (Axon Instruments). For data analysis, idealized traces were created. Open probabilities (Po) were determined by half-amplitude threshold analysis of single-channel recordings as previously described (6). In multichannel experiments, we estimated the global open probability (nPo). In the figures, we show the Po (for single channels) or nPo/x (for multiple channels), with x representing the maximal number of current levels observed.
When indicated, 1–50 µl of halothane stock solution were added to the cis chamber (final halothane levels ranging from 10 µM to 0.5 mM). During the time of the experiments (20–30 min), the loss of halothane from the cis chamber was <10% (estimated using gas chromatography at Carbon Dynamics).
Measurements of Ca2+ uptake/leak by SR microsomes. Ca2+ uptake by SR microsomes was measured with a spectrophotometer (Cory 50, Varian, Walnut Creek, CA) using the Ca2+-sensitive dye antipyrylazo III (APIII). SR membrane vesicles (50 µg/ml) were added to 1 ml phosphate buffer containing (in mM): 100 KH2PO4, 1–7 MgCl2 (free Mg2+ = 0.2–2), 1–4 ATP, and 0.2 APIII; pH 7.0. Ca2+ uptake was initiated by the addition of 30 µM Ca2+ to the medium and measured as changes in absorbance of APIII between 710 and 790 nm. In some experiments, ruthenium red (10 µM) was used to block the Ca2+ leak from SR. In SR vesicles preloaded with Ca2+, we also studied the effects of halothane on the rate of Ca2+ leak after addition of cyclopiazonic acid (20 µM), which inhibits the SR calcium pump [sarco(endo)plasmic reticulum Ca2+-ATPase, SERCA]. The Ca2+ efflux from the vesicles is mainly via the ryanodine receptors as indicated by its block by ruthenium red (10 µM).
Statistical analysis. Data are presented as means ± SE of n measurements. Statistical comparisons between groups were performed with Student's t-test of paired differences. Differences were considered statistically significant at P < 0.05.
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RESULTS |
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4 pA) upward deflections of the current. The cytosolic free Ca2+ concentration was kept at 2 µM. As previously reported (6), in the absence of Mg2+-ATP, skeletal RyR1 channels have low or moderate activity. Under these conditions, cumulative increase of halothane levels (0.12 to 0.5 mM) in the cytosolic bathing solution had no significant effect on RyR1 activity (Fig. 1, A and C, open triangles). However, in the presence of Mg2+-ATP (4 mM Mg2+/5 mM ATP; free Mg2+ = 0.25 mM), halothane induced a marked increase in RyR1 activity (Fig. 1B). Po increased from 0.13 ± 0.09 (control) to 0.38 ± 0.11 with 0.125 mM halothane, to 0.48 ± 0.14 with 0.25 mM halothane and to 0.62 ± 0.12 with 0.5 mM halothane (Fig. 1C, closed triangles).
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100 µM, regardless of the presence/absence of Mg2+-ATP (Fig. 2, A, closed circles and B, closed circles). In the absence of Mg2+-ATP, halothane had no effect on channel activity (Fig. 2A). In the presence of Mg2+-ATP, halothane-induced activation of RyR1 was observed when cytosolic free Ca2+ levels were higher than 2 µM (Fig. 2B). Our results suggest that RyR1 sensitivity to halothane not only depends on the presence of Mg2+-ATP but also on the cytosolic Ca2+ levels.
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As shown in Fig. 4, halothane-induced activation of RyR1 was not affected by addition of 20 µM dantrolene. Under control conditions (2 µM Ca2+, presence of Mg2+-ATP), RyR1 had low or moderate activity (Po = 0.05 ± 0.02). Halothane (0.5 mM) increased RyR1 activity to Po = 0.33 ± 0.05. Subsequent addition of 20 µM dantrolene did not change RyR1 activity (Po = 0.36 ± 0.09, n = 8). Dantrolene, at its limit of solubility (33 µM), was also found without effects on the activity of halothane-activated RyR1 (Po = 0.32 ± 0.07; n = 8).
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Halothane increases activity of cardiac ryanodine receptors (RyR2) in the absence of Mg2+-ATP. Single-channel experiments were performed using 50 mM lumenal Ca2+ as the current carrier and in the absence of Mg2+-ATP. Figure 5 shows examples of multiple channel recordings before (Fig. 5A) and after (Fig. 5B) addition of 0.5 mM halothane to the cytosolic chamber. Halothane (0.5 mM) increased cardiac ryanodine receptor activity at resting (100 nM) cytosolic free Ca2+ levels (Fig. 5, a1 vs. b1) as well as at activating (1 µM) cytosolic free Ca2+ levels (Fig. 5, a2 vs. b2). The average Po values are shown in Fig. 5C. We also found that halothane was an effective agonist of cardiac RyR2 in the presence of Mg2+-ATP (results not shown).
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33 µM dantrolene, respectively; n = 8). In the absence of halothane, dantrolene (20 µM) was also without effect on the activity of RyR2 activated by 5 µM Ca2+ (Po = 0.39 ± 0.07 and 0.40 ± 0.07 before and after addition of dantrolene, respectively; n = 6). Halothane affects SR Ca2+ uptake and leak in skeletal muscle SR microsomes. The rate of Ca2+ uptake by isolated skeletal SR microsomes is a very sensitive macroscopic measurement to test modulation of both RyR1 and SERCA activity. This method, which evaluates populations of RyR1s, was used here to confirm our results of halothane action in planar bilayers. Figure 6A shows the time course of Ca2+ uptake into SR microsomes in response to Ca2+ spikes under control conditions as well as for SR microsomes incubated with halothane and halothane + ruthenium red. After Ca2+ is added, there is rapid uptake. The net Ca2+ uptake equals the SR Ca2+ influx (mediated by SERCA) minus the Ca2+ leak rate from the SR vesicles (via RyRs). In the experiments conducted in the presence of ruthenium red (10 µM), where the RyR-mediated Ca2+ leak was fully inhibited, the time courses of Ca2+ uptake were not affected by halothane (Fig. 6B, open vs. closed triangles and Fig. 6C, striped vs. stipled bars; n = 4 paired experiments). This suggests that halothane does not affect SERCA-mediated Ca2+ uptake.
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A more direct estimation of macroscopic RyR1 activity can be obtained by loading the skeletal muscle SR microsomes with Ca2+ and measuring its release (or leak) after addition of cyclopiazonic acid (an inhibitor of SERCA). Figure 7A shows the time course of Ca2+ release from SR microsomes after addition of cyclopiazonic acid. Notice the large increase in Ca2+ leak from the SR microsomes incubated with halothane. In contrast, in the presence of ruthenium red, no Ca2+ leak was observed under control conditions or after incubation with halothane. This indicates that in our conditions Ca2+ leak was entirely mediated by RyRs. As shown in Fig. 7B, the effect of halothane on SR Ca2+ leak was highly dependent on ATP and Mg2+ concentrations (n = 5 paired experiments). Maximal halothane-induced activation of Ca2+ release was observed with 4 mM ATP (Fig. 7B, open circles) at 0.25 mM free Mg2+.
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Thus, the results obtained with skeletal muscle SR microsomes are in agreement with the observations using RyR1 reconstituted in lipid bilayers. These results confirm that halothane action is synergized by high ATP-low Mg2+ in the SR bathing solution as well as by increasing SR lumenal Ca2+ load. In addition, the SR Ca2+ release studies also indicate that dantrolene may not directly affect RyR1 in native SR microsomes.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Our results indicate that halothane sensitivity of RyR-mediated Ca2+ release in skeletal fibers, but not in cardiac myocytes, would heavily depend on the cellular levels of endogenous modulators (Mg2+, ATP, and Ca2+). They also suggest that dantrolene inhibition of RyR-mediated Ca2+ release reported in cells may result from an "indirect" action on RyRs.
Endogenous agonists affect halothane-induced activation of RyR1. It has been shown that halothane stimulates contraction and SR Ca2+ release from normal skeletal muscle (26, 33). Here, we demonstrate that halothane induces an increase in the activity of skeletal RyR1 channels taken from wild-type rabbit skeletal muscle. In a previous report, halothane has been shown to be without effect on skeletal RyR1 taken from wild-type animals reconstituted in lipid planar bilayers (28). This apparent discrepancy, however, can be explained by the differences in experimental conditions. Indeed, we found that the halothane efficacy as an RyR1 activator was highly dependent on the presence of certain cellular cytosolic modulators (i.e., Ca2+, Mg2+, and ATP) as well as on SR Ca2+ load. Early reports were carried out using simple cytosolic solutions, which did not contain Mg2+-ATP and low Ca2+ (10–100 µM) in the lumenal solutions (28). We also found no effects of halothane under similar conditions.
In all the conditions tested, cytosolic ATP was required to obtain halothane-induced increase in RyR1 activity. Calcium was also a limiting component, since no effect of halothane was observed at cytosolic resting Ca2+ levels (i.e., when the channels are mainly closed). Any condition that favors an increase in RyR1 activity (decrease in [Mg2+]cyt or increase in [Ca2+]cyt) made the channels more susceptible to activation by halothane. These results are consistent with a previous report, where 1 mM halothane-induced SR Ca2+ release in normal skeletal muscle skinned fibers only at low (0.1–0.4 mM) Mg2+ (12).
Possible mechanism of action of halothane on RyR1 activity. The present channel studies indicate that the RyR1 must be, at least minimally, active to be sensitive to halothane since no effect of halothane was observed when the channels were closed (i.e., at resting [Ca2+]cyt, high [Mg2+]cyt, or in the absence of ATP). Accordingly, our SR Ca2+ loading/release assays suggest that the action of halothane requires a moderate level of Ca2+ leak, as induced by high lumenal Ca2+ levels as well as high ATP-low Mg2+ in the cytosol.
It is known that RyR1 is an ATP-gated channel (7, 14, 41). ATP binding greatly increases the activity of skeletal RyR1, including those channels that display low open probabilities (Po 0.01) in the presence of maximal activating cytosolic Ca2+ levels (7). On the other hand, Mg2+ is an inhibitor of RyR1 activity. Mg2+ interferes with Ca2+ binding to cytosolic high affinity (micromolar) divalent cation binding sites that activate the channel (10, 14). In addition, Mg2+ binds to low-affinity (millimolar) inhibitory sites (10, 14). Thus, in the conditions that favored halothane action, the high-affinity (activating) binding sites in RyR1 were partially occupied by Ca2+. Furthermore, high levels of ATP contributed to maintain RyR1 in a high-activity mode.
The halothane-counteracting action of Mg2+ can be explained by decreased occupancy by Ca2+ of high-affinity (activating) sites, increased occupancy by Mg2+ of low-affinity (inhibitory) sites, and formation of Mg-ATP complexes (decrease in free ATP levels). All these actions will greatly decrease RyR1 activity (7, 14).
A previous report showed that halothane induces little or no Ca2+ release from SR membrane vesicles if the Ca2+ content in the SR vesicles is below a threshold level suggesting that lumenal Ca2+ fundamentally affect the action of halothane (29). Likewise, we found that halothane action was dependent on lumenal Ca2+ levels. High lumenal Ca2+ increases RyR1 open probability and sensitivity to ATP (14, 40) and appears to decrease binding of divalent ions to cytosolic low affinity sites (16).
Taken together, our results suggest that halothane binding per se is not enough to activate quiescent RyR1 channels but appears to stabilize the open state of partially activated channels. To be effective, halothane requires the interaction of the RyR1 channel with physiological modulators. Thus, studies on the modulation of isolated RyR1 channels by halothane-like agents should be conducted in the presence of these modulators to get a better comparison with the effects of those agents on RyR1-mediated calcium release in the cellular environment.
Pathophysiological implications: malignant hyperthermia. Our main observation is that the action of halothane (and possibly halothane-like anesthetics) on skeletal RyR1-mediated Ca2+ release would heavily depend on the cellular levels of endogenous agonists. These results may have importance to explain the variability in the contracture tests that are utilized in the diagnosis of MH susceptibility. Although the RyR1 mutations per se could be the main cause for the hypersensitivity to halothane observed in MH-susceptible individuals, it is also plausible that, in some cases, changes in the channel environment might synergize the action of halothane. In this regard, increased resting cytosolic Ca2+ levels have been found in cells carrying some MH mutations (18, 24, 31, 46, 47). This increase in cytosolic Ca2+ correlated with higher resting activity of mutated RyR1 channels as detected using [3H]ryanodine binding (47, 48). Increased RyR1 resting activity would make the channels more susceptible to halothane effects. Notice, also, that increased resting RyR1 activity could also result from diminished negative control by dihydropyridine receptor (DHPR) L-type calcium channels (36, 52).
It is known that RyR1 carrying MH mutations are less sensitive to Mg2+ inhibition (32, 42). Decreased Mg2+ sensitivity would result in higher RyR1 basal activity, and thus, higher sensitivity to halothane. Increased RyR1 basal activity would also be found with decreased cytosolic Mg2+ levels, again increasing RyR1 sensitivity to halothane. In this regard, MH susceptibility has been associated with increased insulin levels (8), which are known to decrease intracellular Mg2+ in muscle (22).
It is known that ATP levels could largely change in skeletal fibers depending on fiber type and cellular metabolism (19). This would greatly affect the activity of ATP-gated RyR1 and consequently, change its sensitivity to halothane-induced activation. If a decrease in ATP levels occurs during manipulation of skeletal fibers under in vitro conditions (1, 30, 44), the basal activity of RyR1 would decrease and thus the response to halothane could be significantly attenuated, originating false negatives in MH-susceptibility tests.
RyR1 channels are modulated by lumenal Ca2+ levels (40). Accordingly, we show that halothane-induced SR Ca2+ release is enhanced when the SR Ca2+ content is increased. It has been reported that the response to the agonist caffeine of skeletal fibers from MH-susceptible individuals is less sensitive to decreasing lumenal Ca2+ (11). Moreover, MH-susceptible pigs display halothane-induced Ca2+ release at much lower levels of Ca2+ content than normal pigs (29). Therefore, we would expect an increased response to halothane when comparing MH-susceptible muscle with normal muscle with equivalent SR Ca2+ load. Notice that increases in SR Ca2+ load could also enhance halothane-induced RyR1 activation.
In summary, the increased sensitivity to halothane observed in MH-susceptible skeletal fibers could be due to mutations in the RyR1 that increase its binding affinity for volatile anesthetics, or mutations that change RyR1 sensitivity to cytosolic modulators (Ca2+, ATP, and Mg2+), or to SR Ca2+ load. Our results also suggest that environmental changes in levels of these modulators (either induced by pharmacological agents or due to pathologies) could make the wild-type RyR1 channels more susceptible to halothane.
Modulation of cardiac RyR2 activity mediated by halothane. Halothane has been reported to increase Ca2+ release in isolated myocytes (45). Halothane was also found to be effective on chemically skinned myocardium, where its action on ruthenium red-sensitive Ca2+ release did not require the presence of nucleotides or relatively high [Ca2+] (17). Accordingly, we show that halothane has a robust activating effect on RyR2 regardless of the presence/absence of Mg2+-ATP. Previous studies also found that halothane is able to activate cardiac RyR2 with Cs+ as current carrier; i.e., with low levels of lumenal Ca2+ (5). Thus our studies and previous reports consistently show that halothane-induced activation of cardiac RyR2 is much less sensitive to the channel environment than halothane-induced activation of skeletal RyR1 isoform.
Lack of effect of dantrolene on halothane-induced activation of RyRs. The muscle relaxant dantrolene has been shown to prevent halothane effects both in MH-susceptible individuals and experimental animal models (21, 27, 47). Dantrolene has also been found to improve intracellular Ca2+ handling in hearts (25). It has been well established that the skeletal RyR1 and cardiac RyR2 are direct targets of dantrolene, as revealed by biochemical photoaffinity labeling experiments (34, 35). Dantrolene binding appears to stabilize interactions between domains in the RyR1 molecule believed to be important for the functional regulation of the channel (20). However, the results shown in the present work indicate that dantrolene action may not be related to direct inhibition of RyR channels.
Our results in cardiac RyR2 are in agreement with previous reports (15). On the other hand, previous studies on the effect of dantrolene on skeletal muscle RyR1 lead to inconsistent results. Early reports suggested that nanomolar levels of dantrolene activate skeletal RyR1, whereas micromolar levels inhibit the channels (27). Differently, [3H]ryanodine-binding studies only found partial inhibitory effects with micromolar levels of dantrolene (13, 15). Partial inhibition by dantrolene of tymol-induced SR Ca2+ release was also found (43). In that report, as well as in a recent communication (4), no effect of dantrolene was observed on RyR1 function studied in bilayers, which is in agreement with the present work.
The cause of the discrepancies among studies of dantrolene action on RyR1 channels is unclear. Possible candidates could include differences in the experimental conditions (e.g., Mg2+-ATP, lumenal and cytosolic Ca2+), which may affect dantrolene efficacy. The method utilized for the isolation of SR microsomes may also introduce variability in the results. In skeletal fibers, L-type Ca2+ channels (DHPR) in t-tubular membranes are a key component of excitation-contraction coupling as they modulate RyR1 through physical interactions (14). In "triad-like" SR preparations but not in "terminal cisternae-like" preparations used here (39), those t-tubular structures containing DHPR remain associated and may modulate RyR1 in SR membranes. Thus, the action of dantrolene on RyR1-mediated Ca2+ release observed in cells (43, 47) could reflect changes in these DHPR-RyR1 interactions. Indeed, dantrolene was found to inhibit the binding of dihydropyridine analogs to DHPR more effectively than the binding of [3H]ryanodine to RyR1 (13). Dantrolene was also found to inhibit DHPR Ca2+ currents (4, 43). In addition, azumolene (a dantrolene analog) decreased the frequency but not the shape (amplitude, duration, and width) of local events of RyR1-mediated Ca2+ release (Ca2+ sparks), indicating that the gating of RyR1 channels during the events was not affected by dantrolene derivatives (49). Similar decrease in Ca2+ spark frequency is found with the DHPR antagonist nifedipine, which appears to stabilize physical interactions between DHPR and RyR1 (52).
Therefore, it is possible that the action of dantrolene on skeletal muscle and heart, including therapeutic properties, would reside in its interaction with target molecules other than RyRs. As indicated, a possible candidate would be the DHPR. However, various alternative dantrolene targets have been suggested in other studies, including the inhibition of RyR1-coupled store-operated calcium entry (50). Still, we cannot rule out the possibility that RyR1 is a direct target for dantrolene action but that the RyR1-dantrolene interaction requires the presence of an ancillary protein that is removed during the vesicle isolation.
In conclusion, the present work shows that the response of RyR1 channels to the volatile anesthetic halotane is highly sensitive to the levels of cytosolic modulators (Ca2+, ATP, and Mg2+) as well as to the SR Ca2+ load. Consequently, these factors represent important variables to be controlled when studying the action of halothane-like anesthetics at the cellular level. In particular, tests used to determine MH susceptibility should take into consideration that variations in the intracellular levels of endogenous RyR1 modulators induced by an abnormal metabolic status of the patients or by changes in the cell environment during the manipulation of the samples would affect the sensitivity of the skeletal fibers to halothane, inducing variability in the outcome of the tests.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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