Effects of Mg2+ and SR luminal Ca2+ on caffeine-induced Ca2+ release in skeletal muscle from humans susceptible to malignant hyperthermia

doi:10.1113/jphysiol.2002.022749

Effects of Mg²⁺ and SR luminal Ca²⁺ on caffeine-induced Ca²⁺ release in skeletal muscle from humans susceptible to malignant hyperthermia

Adrian M. Duke, Philip M. Hopkins* and Derek S. Steele

School of Biomedical Sciences, University of Leeds, Woodhouse Lane, Leeds LS2 9JT and *St James's University Hospital, Leeds LS9 7TF, UK

ABSTRACT

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Abstract
Introduction
Methods
Results
Discussion
References

Regulation of the ryanodine receptor (RYR) by Mg²⁺ and SR luminal Ca²⁺ was studied in mechanically skinned malignant hyperthermia susceptible (MHS) and non-susceptible (MHN) fibres from human vastus medialis. Preparations were perfused with solutions mimicking the intracellular milieu and changes in [Ca²⁺] were detected using fura-2 fluorescence. At 1 mM cytosolic Mg²⁺, MHS fibres had a higher sensitivity to caffeine (2-40 mM) than MHN fibres. The inhibitory effect of Mg²⁺ on caffeine-induced Ca²⁺ release was studied by increasing [Mg²⁺] of the solution containing 40 mM caffeine. Increasing [Mg²⁺] from 1 to 3 mM reduced the amplitude of the caffeine-induced Ca²⁺ transient by 77 ± 7.4 % (n = 8) in MHN fibres. However, the caffeine-induced Ca²⁺ transient decreased by only 24 ± 8.1 % (n = 9) in MHS fibres. In MHN fibres, reducing the Ca²⁺ loading period from 4 to 1 min (at 1 mM Mg²⁺) decreased the fraction of the total sarcoplasmic reticulum (SR) Ca²⁺ content released in response to 40 mM caffeine by 90.4 ± 6.2 % (n = 6). However, in MHS fibres the response was reduced by only 31.2 ± 17.4 % (n = 6) under similar conditions. These results suggest that human malignant hyperthermia (MH) is associated with reduced inhibition of the RYR by (i) cytosolic Mg²⁺ and (ii) SR Ca²⁺ depletion. Both of these effects may contribute to increased sensitivity of the RYR to caffeine and volatile anaesthetics.

(Received 17 April 2002; accepted after revision 1 July 2002; first published online 30 August 2002)
Corresponding author D. S. Steele: School of Biomedical Sciences, University of Leeds, Woodhouse Lane, Leeds LS2 9JT, UK. Email: d.steele{at}leeds.ac.uk

INTRODUCTION

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Abstract
Introduction
Methods
Results
Discussion
References

Malignant hyperthermia (MH) is one of the most likely causes of a serious adverse outcome to general anaesthesia in healthy individuals (for review see Mickelson & Louis, 1996). A genetic predisposition to develop MH is present in 1:8500 of the population and when a reaction occurs, the mortality is ~4 % (Hopkins, 2000). To date, much of the existing functional data relating to MH is derived from experiments on porcine muscle, where the underlying genetic abnormality is an Arg615Cys mutation of the RYR (Fujii et al. 1991). Predisposition to human MH is much more complex; there is both locus and allelic heterogeneity with five susceptibility loci in addition to the ryanodine receptor locus (RYR1) identified (Levitt et al. 1992; Iles et al. 1994; Monnier et al. 1997; Robinson et al. 1997), although RYR1 appears to play a major role in more than 50 % of families (Manning et al. 1998; Brandt et al. 1999). Approximately 30 mutations in RYR1 have been reported (Sambuughin et al. 2001) and all of them lead to an amino acid change except for a deletion in one case.

Despite the genetic heterogeneity of MH, the functional consequences appear remarkably similar. The contracture thresholds for halothane and caffeine are significantly lower in muscle biopsy samples obtained from patients susceptible to MH. This difference in sensitivity to caffeine and halothane forms the basis of the in vitro contracture test (IVCT), which is used to diagnose the condition (European Malignant Hyperpyrexia Group, 1984). A higher sensitivity of the SR Ca²⁺ release mechanism to caffeine or halothane has also been reported in SR vesicles, isolated RYRs incorporated into lipid bilayers and skinned fibre preparations obtained from MHS muscle (Fill et al. 1991; Louis et al. 1992; Adnet et al. 1993).

In addition to the effects of caffeine and volatile anaesthetics, there is evidence that regulation of the RYR by Ca²⁺ and Mg²⁺ is altered in MH. In the presence of low [Mg²⁺], the RYR can be activated by an increase in cytosolic [Ca²⁺] (for review see Endo, 1977). Studies on skinned fibre preparations suggest that the RYR has a higher sensitivity to Ca²⁺ in muscle from MHS patients (Kawana et al. 1992). This is supported by experiments showing that the Ca²⁺ sensitivity of ryanodine binding to isolated SR vesicles is increased in preparations derived from MHS muscle (Valdiva et al. 1991). Furthermore, in skinned skeletal muscle fibres from MHS pigs, the inhibitory effect of Mg²⁺ on RYR was significantly reduced (Owen et al. 1997). This is a potentially important finding, because reduced inhibition by Mg²⁺ might increase the sensitivity of the RYR to activators including halothane and caffeine. However, it has not yet been established whether altered Mg²⁺ regulation of the RYR is a common feature of human MH.

Another factor, which is poorly understood in relation to MH, is the influence of luminal Ca²⁺ on the activation properties of the RYR. Early studies on isolated SR investigated the 'luminal threshold' for SR Ca²⁺ efflux in normal and MHS muscle (Ohnishi et al. 1983). The concept of a luminal threshold was based on the observation that a defined amount of Ca²⁺ could be loaded into SR vesicles, such that any further addition of Ca²⁺ to the medium resulted in Ca²⁺ efflux via the RYR. This effect may be explained by recent observations on isolated channels, showing that luminal Ca²⁺ can increase the sensitivity of the RYR to activation by cytosolic Ca²⁺ (Sitsapesan & Williams, 1995). Interestingly, the luminal threshold for Ca²⁺ efflux via the RYR was significantly lower in SR vesicles from porcine MHS muscle (Ohnishi et al. 1983). However, no such effect was observed in vesicles isolated from human MHS muscle (Fletcher et al. 1993).

The main aim of the present study was to investigate the possibility that inhibition of the RYR by Mg²⁺ may be reduced in human MH. In addition, experiments were carried out to assess whether SR luminal Ca²⁺ has differential effects on RYR regulation in MHN and MHS skeletal muscle fibres. Experiments were carried out on mechanically skinned fibres isolated from vastus medialis muscle, obtained from patients attending for investigation of MH susceptibility. Rapid application of caffeine released Ca²⁺ from the SR, which was detected using fura-2 fluorescence. It was found that the inhibitory influence of Mg²⁺ on caffeine-induced Ca²⁺ release was significantly lower in MHS fibres. Furthermore, in both MHN and MHS muscle, decreasing the Ca²⁺ loading period reduced the fraction of the total SR Ca²⁺ content released by 40 mM caffeine. However, this inhibitory effect of SR Ca²⁺ depletion was less pronounced in MHS fibres. These results suggest that human MH is associated with reduced inhibition of the RYR by cytosolic Mg²⁺and SR Ca²⁺ depletion. Both of these effects may contribute to increased sensitivity of the RYR to caffeine and volatile anaesthetics in human MH.

METHODS

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Abstract
Introduction
Methods
Results
Discussion
References

Solution composition

All chemicals were purchased from Sigma unless otherwise stated. For most experiments a basic solution was prepared containing (mM): Hepes 25, EGTA 0.15, creatine phosphate 10, ATP 5, KCl 100 and Fura-2 1.5. The free [Mg²⁺] and [Ca²⁺] of the basic solution was adjusted to 1 mM and 120 µM, respectively. Two millimolar NaN₃ was added routinely to inhibit mitochondrial activity. Where necessary, a computer program was used to calculate free ion concentrations (REACT, Duncan et al. 1999). Corrections for ionic strength, details of pH measurement, allowance for EGTA purity and the principles of the calculations are described elsewhere (Smith & Miller, 1985). All experiments were done at room temperature (20-22 °C), pH 7.0. In control experiments, potassium propionate or K-HDTA was used in place of KCl. In these experiments, the basic phenomena were similar, although the amplitude of the caffeine-induced fluorescence transient was ~10 % smaller in solutions lacking Cl^-. This is consistent with previous reports that Cl^- has a moderate facilitating effect on SR Ca²⁺ release (Fruen et al. 1996).

Preparation

Samples of vastus medialis muscle were obtained by open biopsy from patients attending for investigation of MH susceptibility at St James's Hospital Leeds, UK. Approximately 1 g of muscle was removed for the IVCT. With institutional Research Ethics Committee approval and informed patient consent, an additional bundle (0.2 g) was used to provide material for studies on mechanically skinned muscle preparations. All procedures were carried out according to the Declaration of Helsinki. The IVCT provided the primary method of categorising MHN and MHS tissue, according to the criteria for MH research of the European MH Group (European Malignant Hyperpyrexia Group, 1984). Patients with equivocal IVCT diagnoses were excluded from the present study. This ensured a high sensitivity and specificity of the MHS diagnosis (98 % and 94 %, respectively; Ording et al. 1997).

The bundle of muscle used to provide skinned fibres was placed in a 'relaxing' solution approximating the intracellular milieu (see above). The muscle sample was pinned on a Sylgard bath and individual muscle fibres were then isolated and mechanically skinned with fine forceps. Vastus medialis is of mixed fibre type and strontium sensitivity tests were routinely carried out to classify the fibres as type 1 or type 2 (Fink et al. 1990). It was found that most of the preparations did not generate significant tension at pSr 5.2, suggesting that the majority of selected fibres were type 2. Any preparations generating significant tension at pSr 5.2 were not used in the present study.

Relative to previous studies on rat fibres under similar conditions, there was increased variation in the quality (by visual inspection) of the human muscle fibres from both MHS and MHN muscle and in the stability of the contractile response. There was also an increased probability of the fibres snapping. This may reflect variations in age/fitness of the patients and possibly aspects of the biopsy procedure. However, there were no apparent differences between MHN and MHS muscle in the contractile responses.

Analysis of the biopsy material for RYR mutations was incomplete and the data have not been included. However, given the genetic heterogeneity of the condition and the fact that the majority of patients were unrelated (see figure legends), it seems likely that multiple RYR mutations are present in the MHS tissue used in this study. Furthermore, despite the fact that ~30 mutations of the RYR have been detected in MHS tissue, in ~75 % of MHS cases none of these known mutations is present (Robinson et al. 2002). This suggests that there may be other unknown mutations and possibly non-genetic factors which predispose to human MH.

Apparatus

The apparatus for simultaneous measurement of force and SR Ca²⁺ release is described in detail elsewhere (Duke & Steele, 1998b). Briefly, a mechanically skinned muscle fibre was mounted in a shallow bath with a coverslip base. The fibre was attached between a fixed support and a force transducer (SensoNor, Norway) using monofilament thread (30 µm diameter, Ethicon Ltd, Edinburgh, UK) within stainless steel tubes (i.d. 100 µm, Goodfellow Cambridge Ltd, Huntington, UK). A cylindrical Perspex column (4 mm in diameter) was lowered to within a few micrometres of the fibre surface. The volume of solution between the coverslip and the column was ~6 µl. Throughout the experimental protocols, preparations were perfused by pumping solution at 0.8 ml min^-1 via a narrow duct (200 µm diameter) passing through the centre of the column. Waste solution was collected continuously at two points at the edge of the column.

Caffeine was applied rapidly using a specially constructed syringe pump, which allowed each of eight channels to be controlled independently via a computer interface. The syringes (5 ml), containing the caffeine solutions, were connected via narrow cannulae (i.d. 0.5 mm) to a series of injection ducts near the base of the column. The experimental bath was placed on the stage of a S200 Nikon Diaphot inverted microscope. The muscle fibre was viewed via a times 40 Fluor objective (Nikon CF Fluor, NA 0.75) and the length was increased to ~20 % above slack length. The preparation was alternately illuminated with light of wavelengths 340 nm and 380 nm at 50 Hz frequency using a spinning wheel spectrophotometer (Cairn Research, Faversham, Kent, UK). The spatially averaged [Ca²⁺] within the visual field containing the preparation was indicated by the ratio of light intensities emitted at > 500 nm. Light emitted from areas of the field not occupied by the muscle image was excluded using a variable rectangular diaphragm on the side port of the microscope.

Rationale for experiments involving rapid application of caffeine

In the present study caffeine was utilised in a number of different ways. A maximal release of SR Ca²⁺ was induced by rapid application of a solution containing 40 mM caffeine, but lacking Mg²⁺. The amplitude of the resulting fura-2 fluorescence transient was used as an index of the SR Ca²⁺ content. The fura-2 signal is mostly derived from the preparation. However, some fluorescence is also collected from fura-2 in the surrounding solution. As it is not practical to quantify accurately the relative contribution of fluorescence collected from the preparation and the surrounding solution in each experiment, the original records are presented as the 340:380 fluorescence ratios and were not converted to [Ca²⁺] (see Duke & Steele, 1998b for discussion). In presentation of cumulative data, the amplitude of fluorescence transients was expressed relative to a maximal response to caffeine/zero Mg²⁺, under standardised loading conditions.

Brief application of caffeine results in a rapid release of Ca²⁺ from the SR. A 500 ms application was sufficient to maximise the amplitude of the resulting fluorescence transient. However, caffeine is then washed away from the preparation, allowing a rapid decline in [Ca²⁺] due to SR Ca²⁺ uptake and some diffusion of Ca²⁺ into the surrounding solution (Duke & Steele, 1998b). The comparatively brief twitch-like responses cause little damage and can be repeated without significant rundown over tens of minutes. As the descending phase of the caffeine-induced fluorescence transient is influenced by SR Ca²⁺ reuptake, the amplitude of the transient has been used as an index of the SR Ca²⁺ content rather than the integral.

In some experiments, the free [Mg²⁺] of the caffeine-containing solution was increased (to 2 mM or 3 mM), while [Mg²⁺] of the basic perfusing solution remained at 1 mM. The object of these experiments was to investigate inhibition of the SR Ca²⁺ release mechanism by Mg²⁺. However, it is necessary to consider whether the increase in [Mg²⁺] may cause an artifactual change in the fura-2 fluorescence ratio. The first consideration is that increasing the free [Mg²⁺] from 1 to 3 mM could increase the free [Ca²⁺] within the bath, due to competition with Ca²⁺ bound to EGTA. Calculations using REACT (see above) suggest that a 2 mM increase in free Mg²⁺ would have a negligible effect at resting [Ca²⁺]. However, previous calculations suggest that the average [Ca²⁺] within the preparation may increase to 700-800 nM Ca²⁺, at the peak of a caffeine-induced Ca²⁺ transient under similar conditions (Duke & Steele, 1998b). At this [Ca²⁺], a 2 mM increase in [Mg²⁺] is calculated to increase the free [Ca²⁺] by ~ 7 %. Such an effect may result in a small reduction in the apparent influence of Mg²⁺ on caffeine-induced Ca²⁺ release.

Another potential difficulty is that Mg²⁺ might compete with Ca²⁺ for binding to fura-2. However, fura-2 was developed specifically to have a high selectivity for Ca²⁺ compared with Mg²⁺. In vitro measurements have shown that the dissociation constant of fura-2 for Mg²⁺ is ~10 mM under conditions relevant to the present study. Furthermore, changes in Mg²⁺ over the range 0-3 mM have been shown to have negligible effect on the spectral properties of fura-2 (see Fig. 4, Grynkiewicz et al. 1985). Therefore, it seems unlikely that the changes in Mg²⁺ used in the present study will have major effects on fura-2 or free Ca²⁺. In any case, such effects are unlikely to have any bearing on the conclusions of the present study, because the same protocols were applied to both MHS and MHN fibres.

Data recording and analysis

In all experiments, the ratio and individual wavelength intensities and isometric force signals were low-pass filtered (-3 db at 30 Hz) and digitised for later analysis using a PC (pentium) with a Data Translation 2801A card. All n values indicate the number of patients and only one fibre was used from each patient. The numbers of MHN and MHS patients are indicated in the figure legends. Significance (P < 0.05) levels were calculated using Student's t test for unpaired observations and the data are presented as means ± 1 standard error of the mean (S.E.M.).

RESULTS

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Abstract
Introduction
Methods
Results
Discussion
References

Effect of loading period on caffeine-induced Ca²⁺ release

Figure 1A shows simultaneous recordings of the 340 nm/ 380 nm fluorescence ratio (upper panel) and isometric force (lower panel) from a mechanically skinned MHS muscle fibre. The preparation was perfused constantly with weakly Ca²⁺-buffered solutions containing 1 mM free Mg²⁺ and 1.5 µM fura-2. The resting fluorescence ratio corresponds to a free [Ca²⁺] within the perfusing solution of 120 nM.

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Figure 1. Relationship between duration of Ca²⁺ loading and SR Ca²⁺ content
A, protocol used to assess the relationship between the duration of Ca²⁺ loading and the amount of Ca²⁺ available for release from the SR in response to application of 40 mM caffeine/zero Mg²⁺. Continuous records of the fluorescence ratio (upper panel) and force (lower panel) obtained from a MHS fibre are shown. A maximal Ca²⁺ release was induced at the indicated intervals (1-4 min) by rapid application of 40 mM caffeine/zero Mg²⁺ (filled arrowheads). B, cumulative data for peak force and fluorescence, expressed as a percentage of the control response, i.e. the steady-state response at 4 min intervals. There was no significant difference between MHS and MHN fibres in the normalised amplitude of the tension or Ca²⁺ transients (P > 0.05). All values are means ± S.E.M. and between 7 and 15 values were averaged to obtain each point. The cumulative data were obtained from a total of 25 patients (15 MHN and 10 MHS), of which 24 were unrelated.

Previous studies have shown that even high levels of caffeine do not fully activate the SR Ca²⁺ release channel in fast twitch fibres at 1 mM free Mg²⁺ (Fryer & Stephenson, 1996). However, a maximal Ca²⁺ release can be achieved by simultaneously applying high levels of caffeine and reducing [Mg²⁺] to micromolar levels (Kabbara & Stephenson, 1994). Therefore, in the present study, the maximum releasable pool of SR Ca²⁺ was estimated by rapid injection of a solution with a high concentration of caffeine (40 mM), but lacking Mg²⁺ (40 mM caffeine/zero Mg²⁺). Measurements using furaptra (Mg²⁺-selective fura-2) showed that [Mg²⁺] actually decreased within the preparation to ~50 µM during the caffeine application. Application of 40 mM caffeine/zero Mg²⁺ for 500 ms was sufficient to produce a maximal increase in the 340:380 fluorescence ratio (Ca²⁺ transient), i.e. a more prolonged application did not increase the amplitude of the Ca²⁺ or force transients (not shown).

In this protocol, caffeine/zero Mg²⁺ was applied briefly at variable intervals in order to assess the relationship between the duration of Ca²⁺ loading and the SR Ca²⁺ content in normal and MHS fibres. Increasing the Ca²⁺ loading period (i.e. the time between caffeine applications) from 1 to 4 min caused a progressive increase in Ca²⁺ and force responses. However, increasing the loading period beyond 4 min did not result in any significant increase in the caffeine-induced Ca²⁺ or force transients. This suggests that the releasable pool of SR Ca²⁺ reached a steady state within ~4 min under these conditions. Figure 1A also shows that brief caffeine application allows many responses to be produced without significant deterioration of muscle function.

Accumulated data showing the relationship between the loading period and the amplitude of caffeine-induced force and Ca²⁺ responses in MHN and MHS fibres are shown in Fig. 1B. All responses have been normalised with respect to maximal releases induced by application of 40 mM caffeine/zero Mg²⁺ at a 4 min loading period. There was no significant difference in the relationship between the Ca²⁺ loading period and normalised responses to 40 mM caffeine/zero Mg²⁺ in MHN and MHS fibres. Furthermore, in both groups, there was no significant increase in caffeine-induced Ca²⁺ release between 4 and 8 min. Unless otherwise stated, in subsequent experiments, caffeine was applied at 4 min intervals.

Concentration dependence of caffeine-induced Ca²⁺ release

The basis of the diagnostic IVCT is that MHS muscle has a higher sensitivity to caffeine and halothane than MHN muscle. The test typically involves exposure of intact muscle fibre bundles obtained during open biopsy to increasing levels of caffeine or halothane. Therefore, unlike the conditions used to assess the maximum releasable pool of SR Ca²⁺ (Fig. 1), the free [Mg²⁺] within the cytosol should be around normal physiological levels (0.8-1 mM). At 1 mM free Mg²⁺, even high levels of caffeine induce a submaximal release of Ca²⁺ from the SR (Fryer & Stephenson, 1996) and the amplitude of the resulting Ca²⁺ transient is sensitive to cytosolic factors, which modulate the gating behaviour of the RYR (Duke & Steele, 1998a).

Figure 2A shows the protocol used to investigate the relationship between [caffeine] and SR Ca²⁺ release in the presence of 1 mM Mg²⁺. Simultaneous records of isometric force (lower panel) and the fura-2 fluorescence ratio (upper panel) from a MHN fibre are shown. Forty millimolar caffeine/zero Mg²⁺ was initially applied at 4 min intervals, resulting in a series of force and Ca²⁺ transients (2 shown). This was done to assess the maximum amount of Ca²⁺ available for release from the SR in each fibre. After a further 4 min loading period, a solution containing 5 mM caffeine and 1 mM free Mg²⁺ (the same [Mg²⁺] as the perfusing solution) was rapidly applied. The amplitudes of the associated Ca²⁺ and force transients were markedly reduced. This was followed by a further two control responses to 40 mM caffeine/zero Mg²⁺. The protocol was repeated at a range of [caffeine].

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Figure 2. Concentration dependence of caffeine-induced Ca²⁺ release
A, simultaneous records of 340/380 nm fluorescence ratio and isometric force showing the protocol used to assess the concentration dependence of caffeine-induced Ca²⁺ release. In this example, a MHN fibre was perfused with solution mimicking the cytosol with a free [Mg²⁺] of 1 mM. Forty millimolar caffeine/zero Mg²⁺ was applied briefly at 4 min intervals (filled arrowheads) to establish steady-state Ca²⁺ and force responses. Five millimolar caffeine/1 mM Mg²⁺ was a less effective stimulus for SR Ca²⁺ release. B, accumulated data from MHN and MHS fibres obtained using the protocol shown in Fig. 2A. The response at each concentration of caffeine (1 mM Mg²⁺) is expressed relative to a maximal response to 40 mM caffeine/zero Mg²⁺. All values are means ± S.E.M. and between 5 and 18 values were averaged to obtain each point. The cumulative data were obtained from a total of 31 patients (18 MHN and 13 MHS), of which 28 were unrelated. ** Significant difference between MHS and MHN fibres (P < 0.05).

Figure 2B shows accumulated data obtained using this protocol in MHS and MHN fibres. The amplitude of responses to each [caffeine] (2-40 mM) is expressed relative to the transients resulting from maximal Ca²⁺ release induced by application of 40 mM caffeine/zero Mg²⁺. These results show that caffeine-induced Ca²⁺ release is concentration dependent in both MHN and MHS fibres. However, under these conditions, there is a markedly higher sensitivity to caffeine in MHS fibres.

Effects of [Mg²⁺] on caffeine-induced Ca²⁺ release

Figure 3A shows the protocol used to investigate the effects of Mg²⁺ on caffeine-induced Ca²⁺ release. In this example, a MHN fibre was briefly exposed to 40 mM caffeine/zero Mg²⁺ at 4 min intervals. This produced a series of maximal caffeine-induced Ca²⁺ (upper panel) and force (lower panel) transients (2 shown). After a further 4 min loading period, a solution containing 40 mM caffeine and 3 mM Mg²⁺ was applied. The resulting Ca²⁺ transient was markedly reduced in amplitude. As [Mg²⁺] was only increased in the solution containing caffeine (and not in the perfusing solution) the decrease in Ca²⁺ release reflects an inhibitory influence of Mg²⁺ on the RYR, rather than an indirect effect on Ca²⁺ uptake. On subsequent application of 40 mM caffeine/zero Mg²⁺, the Ca²⁺ and force transients returned to control levels. This protocol was repeated in MHS and MHN fibres at a range of [Mg²⁺]. Figure 3B shows representative caffeine-induced Ca²⁺ transients obtained at 1, 2 and 3 mM Mg²⁺ in a MHN (left) and a MHS (right) fibre. Each transient is superimposed on a preceding control response to 40 mM caffeine/zero Mg²⁺. At each [Mg²⁺], the decrease in the amplitude of the caffeine-induced Ca²⁺ transient was less pronounced in the MHS fibre.

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Figure 3. Effects of [Mg²⁺] on caffeine-induced Ca²⁺ release
A, simultaneous records of 340/380 nm fluorescence ratio and isometric force showing the protocol used to assess the effects of [Mg²⁺] on caffeine-induced Ca²⁺ release. In this example, a MHN fibre was perfused with a solution mimicking the cytosol, with a free [Mg²⁺] of 1 mM. Forty millimolar caffeine/zero Mg²⁺ was applied briefly at 4 min intervals to establish maximal steady-state Ca²⁺ and force responses. Increasing the free [Mg²⁺] in the 40 mM caffeine solution to 3 mM caused a marked reduction in caffeine-induced Ca²⁺ release. B, selected steady-state Ca²⁺ transients from a MHS and a MHN fibre showing the effects of 40 mM caffeine applied in the presence of 1 mM ( filled circle ), 2 mM ( circle ) or 3 mM ( filled square ) Mg²⁺. Each response is superimposed on the preceding maximal release in response to application of 40 mM caffeine/zero Mg²⁺. C, cumulative data using the protocol shown in Fig. 3A. All values are means ± S.E.M. and between 7 and 18 values were averaged to obtain each point. The cumulative data were obtained from a total of 30 patients (18 MHN and 12 MHS) , of which 26 were unrelated. ** Significant difference between MHS and MHN fibres (P < 0.05).

Cumulative data showing the relationship between [Mg²⁺] and the amplitude of the caffeine-induced responses in MHN and MHS fibres are shown in Fig. 3C. All values are expressed as a percentage of the preceding maximal responses to 40 mM caffeine/zero Mg²⁺. Increasing the free [Mg²⁺] in the caffeine solution had a clear inhibitory effect on the amplitude of the Ca²⁺ transients in both groups. However, the inhibitory effect of Mg²⁺ was significantly greater in MHN fibres.

Effect of the SR Ca²⁺ load on caffeine sensitivity

In previous experiments in this study, the effects of caffeine and Mg²⁺ on SR Ca²⁺ release were assessed when the SR was at or near maximal loading capacity (i.e. after a 4 min loading period, Fig. 1). Therefore, the protocol shown in Fig. 4A was designed to investigate whether the SR Ca²⁺ content affects the sensitivity of the RYR to caffeine in MHN (left panel) and MHS (right panel) fibres. In each panel, two maximal responses to 40 mM caffeine/ zero Mg²⁺ were initially induced at 8 min intervals (filled arrowheads). After a further 8 min Ca²⁺ loading period, 40 mM caffeine/1 mM Mg²⁺ was applied (open arrowhead). This protocol was then repeated, but the loading period between each response (to 40 mM caffeine/zero Mg²⁺ or 40 mM caffeine/1 mM Mg²⁺) was reduced to 4, 2 and finally 1 min intervals. For each given loading period, the maximal responses to 40 mM caffeine/zero Mg²⁺ provided an index of the amount of Ca²⁺ available for release from the SR, while the response to 40 mM caffeine (1 mM Mg²⁺) was submaximal, due to the presence of physiological levels of Mg²⁺.

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Figure 4. Effect of the SR Ca²⁺ load on caffeine sensitivity
A, simultaneous records of 340/380 nm fluorescence ratio and isometric force showing the protocol used to assess the effects of the Ca²⁺ loading period on the sensitivity of the caffeine release mechanism. Following a series of maximal steady-state Ca²⁺ and force responses to 40 mM caffeine/zero Mg²⁺, a solution containing 40 mM caffeine/1 mM Mg²⁺ was applied. This was repeated at 8, 4, 2 and 1 min loading periods in MHN (left) and MHS (right) fibres. Filled triangles indicate application of 40 mM caffeine/zero Mg²⁺, open triangles indicate application of 40 mM caffeine, 1 mM Mg²⁺. B, cumulative data showing the relationship between the amplitude of the caffeine-induced Ca²⁺ transient and the duration of SR Ca²⁺ loading in MHS and MHN fibres. Responses are expressed relative to a maximal response to caffeine/zero Mg²⁺ at each loading period. All values are means ± S.E.M., and between 5 and 7 values were averaged to obtain each point. The cumulative data were obtained from a total of 16 patients (9 MHN and 7 MHS), of which all 16 were unrelated. ** Significant difference between MHS and MHN fibres (P < 0.05); n.s., no significant difference.

These records show that in MHN fibres, the fraction of the SR Ca²⁺ content released by 40 mM caffeine in the presence of 1 mM Mg²⁺ decreased markedly as the Ca²⁺ loading period was progressively reduced (left panel). Indeed, when the Ca²⁺ loading period was decreased to 1 min, 40 mM caffeine failed to induce a detectable release of Ca²⁺. This occurred despite the fact that the amplitude of the maximal Ca²⁺ transient induced by 40 mM caffeine/zero Mg²⁺ had only decreased by ~50 %, indicating that a substantial amount of Ca²⁺ remained within the SR. In MHS fibres, the difference between the maximal responses (to 40 mM caffeine/zero Mg²⁺) and the response to 40 mM caffeine/1 mM Mg²⁺ was less pronounced. Furthermore, in MHS fibres, reducing the loading period to 1 min intervals did not abolish Ca²⁺ release in response to 40 mM caffeine/1 mM Mg²⁺.

Cumulative data obtained using this protocol are shown in Fig. 4B. All responses to 40 mM caffeine/1 mM Mg²⁺ are expressed as a percentage of the preceding maximal responses to 40 mM caffeine/zero Mg²⁺. The results show that in MHN fibres, reducing the Ca²⁺ loading period below 4 min markedly decreases the fraction of the total SR Ca²⁺ content released in response to 40 mM caffeine/ 1 mM Mg²⁺. This desensitizing effect of reduced luminal Ca²⁺ was less pronounced in MHS fibres.

DISCUSSION

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Abstract
Introduction
Methods
Results
Discussion
References

SR Ca²⁺ uptake and caffeine sensitivity in MHN and MHS fibres

In both MHS and MHN fibres the SR Ca²⁺ content (assessed by application of 40 mM caffeine/zero Mg²⁺) approached a maximum level within ~4 min of loading (Fig. 1). Increasing the Ca²⁺ loading period to 8 min produced only a small further rise in the amplitude of the Ca²⁺ transient, which was not statistically significant. The relationship between the duration of Ca²⁺ loading and the normalised amplitude of the caffeine-induced Ca²⁺ response appeared similar in MHS and MHN fibres (Fig. 1B). This is consistent with previous studies suggesting that the SR Ca²⁺-ATPase is unaffected in human MH (Nelson, 1988). However, the absence of any difference in Ca²⁺ loading characteristics also suggests that any difference in the resting Ca²⁺ leak via the RYR (see below) is insufficient to affect net Ca²⁺ uptake.

Effects of Mg²⁺ on caffeine-induced Ca²⁺ release in normal and MHS fibres

Studies on isolated RYRs have shown that Mg²⁺ exerts a dual inhibitory effect on the RYR by (i) competing with Ca²⁺ at the activation site and (ii) binding to a low affinity Mg²⁺ inhibitory site (Meissner et al. 1986; Laver et al. 1997). Furthermore, experiments on skinned muscle preparations have led to the suggestion that the physiological Ca²⁺-release process involves a decrease in the affinity of the Mg²⁺ binding site, triggered by interaction between the dihydropyridine receptor (DHPR) and the RYR (Lamb & Stephenson, 1994). In skeletal muscle, cytoplasmic ATP can activate RYR even if the Ca²⁺ activation site is not occupied (Smith et al. 1986). This may explain why reduced Mg²⁺ inhibition, during the physiological activation process, can induce RYR activation at resting levels of Ca²⁺.

Given the central role of Mg²⁺ in the excitation- contraction coupling process, any factor which influences the degree of inhibition of the RYR by cytosolic Mg²⁺ might be expected to have a major influence on SR Ca²⁺ release. However, the consequences of reduced Mg²⁺ inhibition depend on the stimulus needed for RYR activation. A previous study on porcine muscle examined the inhibitory effects of cytosolic Mg²⁺ on the RYR in resting MHS and MHN fibres (Owen et al. 1997). The cytosolic [Mg²⁺] was progressively reduced below normal physiological levels, until Ca²⁺ efflux from the SR occurred via the RYR. In MHS fibres, a smaller decrease in [Mg²⁺] was required to induce SR Ca²⁺ release, suggesting that the inhibitory effect of Mg²⁺ on the RYR is reduced. However, even in MHS fibres it was necessary to reduce the [Mg²⁺] to ~0.2 mM before Ca²⁺ release was apparent. Therefore, in porcine MHS fibres, inhibition of the RYR by normal levels of cytosolic Mg²⁺ appears sufficient to prevent a major loss of SR Ca²⁺ under resting conditions. This may also explain why net Ca²⁺ uptake is similar in MHS and MHN fibres at normal cytosolic levels of Ca²⁺ (Fig. 1).

Previous studies have shown that the twitch and tetanus characteristics of MHN and MHS fibres are similar (for review see Mickelson & Louis, 1996). Therefore, when the DHPR interacts with the RYR, the decrease in Mg²⁺ inhibition may be large enough to obscure any differences in Mg²⁺ sensitivity. In contrast, differences in Mg²⁺ inhibition of RYR are of major importance in determining the amount of Ca²⁺ released from the SR in response to caffeine. In mammalian skeletal muscle fibres, SR Ca²⁺ release is submaximal when very high levels of caffeine (30-40 mM) are applied at normal [Mg²⁺]_i (Fryer & Stephenson, 1996). This is presumably because (in the absence of DHPR activation) caffeine must overcome inhibition of the RYR by cytosolic Mg²⁺. In these circumstances, any intrinsic difference in the degree of RYR inhibition by Mg²⁺ would be expected to alter the sensitivity of the RYR to caffeine. As previously considered, this effect is likely to contribute to the increased sensitivity of MHS fibres to caffeine or halothane in porcine fibres (Owen et al. 1997). In the present study we have shown that Mg²⁺ is less effective at inhibiting caffeine-induced activation of the RYR in MHS fibres isolated from human muscle (Fig. 3). Therefore, despite the genetic heterogeneity of human MH, it appears that reduced inhibition of the RYR by Mg²⁺ is a common feature of the condition.

Effects of SR luminal Ca²⁺ on caffeine-induced Ca²⁺ release

Previous work on mechanically skinned fibres from rat fast (EDL) and slow (soleus) muscle has shown that the caffeine sensitivity of the SR Ca²⁺ release mechanism is dependent on the relative level of Ca²⁺ loading in the SR (Lamb et al. 2001). Under conditions designed to produce an endogenous level of Ca²⁺ loading for each fibre type, slow fibres were significantly more sensitive to caffeine than fast fibres. However, the endogenous SR Ca²⁺ content in soleus fibres is higher than that in EDL fibres. When the two fibre types were Ca²⁺ loaded to the same extent, the difference in caffeine sensitivity was largely abolished.

The luminal dependence of RYR sensitivity is not fully understood. It is possible that the RYR may be influenced by a local increase in [Ca²⁺] resulting from a 'leak' of Ca²⁺ from the SR (Xu & Meissner, 1998). Such an effect would be expected to increase as the SR Ca²⁺ content rises, giving an apparent luminal dependence for RYR activation. However, recent work has shown that exposure of the trans face of the channel to trypsin abolishes luminal regulation of the cardiac RYR by Ca²⁺ (Ching et al. 2000). This suggests that occupation of low affinity Ca²⁺ regulatory sites on the luminal side of the RYR may be responsible for the reported increase in channel sensitivity to cytosolic activators.

The dependence of RYR activation on luminal Ca²⁺ may explain experiments on isolated SR showing that, above a threshold Ca²⁺ content, a RYR-mediated Ca²⁺ efflux pathway is activated (Ohnishi et al. 1983). Of particular relevance to the present study, the luminal threshold of RYR was lower in SR vesicles derived from the porcine model of MH. However, no such effect was detected in vesicles derived from human MHS muscle (Fletcher et al. 1993). This apparent discrepancy between studies on pig and human fibres may reflect methodological differences or complications associated with accumulation of free fatty acids during preparation of SR vesicles (Fletcher et al. 1991). However, in the present study we have shown that in skinned fibres from human MHN and MHS muscle, the sensitivity to caffeine exhibits a marked dependence on the SR Ca²⁺ content (Fig. 4). Decreasing the SR Ca²⁺ loading period reduced the fraction of the total SR Ca²⁺ content released by 40 mM caffeine/1 mM Mg²⁺. Indeed, in MHN fibres, caffeine-induced Ca²⁺ release (at 1 mM Mg²⁺) was effectively abolished by decreasing the Ca²⁺ loading period to 1 min. This contrasts with the situation in MHS fibres where the response to 40 mM caffeine/1 mM Mg²⁺ was near maximal with loading periods from 2-8 min and a substantial release was initiated following 1 min loading.

It is possible that the apparent difference in luminal regulation of the RYR in MH could be a primary pathological mechanism. However, the sensitivity of the RYR to caffeine presumably reflects the combined effects of 'luminal feedback' and the influence of cytosolic channel modulators. Therefore, the fact that SR Ca²⁺ release is inhibited less in MHS fibres when the SR Ca²⁺ content declines could be an indirect consequence of reduced Mg²⁺ inhibition, or possibly increased Ca²⁺ sensitivity. For example, Fig. 3 shows that relative to a maximal release (to 40 mM caffeine/zero Mg²⁺), the response to 40 mM caffeine, 1 mM Mg²⁺ is ~15-20 % smaller in MHN fibres than MHS fibres. Therefore, reduced inhibition by Mg²⁺ will contribute to, or may even explain, the difference between MHN and MHS fibres shown in Fig. 4, at 4 min loading periods. However, the difference between MHS and MHN fibres gets progressively bigger as the loading period is reduced to 2 min and then 1 min. Indeed at a 1 min loading period, the responses in MHN fibres are reduced by ~90 % (relative to a maximal response), whereas in the MHS fibres the responses were reduced by only ~ 30 %. For this to be explained solely in terms of differences in Mg²⁺ inhibition, Mg²⁺ would need to have a proportionally bigger effect at lower SR Ca²⁺ contents. Alternatively, some factor other than Mg²⁺ sensitivity may be involved in the apparent luminal dependence of caffeine, although it is not possible to establish this unequivocally using these methods.

Possible physiological significance

In MHS muscle, reduced inhibition of the RYR by cytosolic Mg²⁺ and possibly increased 'feedback' from SR luminal Ca²⁺ may contribute to increased caffeine and halothane sensitivity. Furthermore, recent studies on normal muscle have highlighted a number of factors which may inhibit SR Ca²⁺ release in the final stages of fatiguing stimulation. This includes ATP depletion, increased cytosolic [Mg²⁺] and reduced SR luminal Ca²⁺ (Blazev & Lamb, 1999; Kabbara & Allen, 2001). Assuming the sustained contractile activity during a MH episode results in a similar pattern of intracellular changes, then raised [Mg²⁺] and SR Ca²⁺ depletion may be less effective at inhibiting SR Ca²⁺ release.

REFERENCES

Top
Abstract
Introduction
Methods
Results
Discussion
References

ADNET, P. J., BROMBERG, N. L., HAUDECOEUR, G., KRIVOSIC, I., ADAMANTIDIS, H., REYFORD, H. & DUPUIS, B. A. (1993). Fibre-type caffeine-sensitivities in skinned muscle fibres from humans susceptible to malignant hyperthermia. Anesthesiology 78, 168-177 [Medline]

BLAZEV, R. & LAMB, G. D. (1999). Low [ATP] and elevated [Mg²⁺] reduce depolarization-induced Ca²⁺ release in rat skinned skeletal muscle fibres. Journal of Physiology 520, 203-215 [Abstract/Full Text]

BRANDT, A., SCHLEITHOFF, L., JURKAT-ROTT, K., KLINGER, W., BAUR, C. & LEHMANN-HORN, F. (1999). Screening of the ryanodine receptor gene in 105 malignant hyperthermia families: novel mutations and concordance with the in vitro contracture test. Human Molecular Genetics 8, 2055-2062 [Abstract/Full Text]

CHING, L. L., WILLIAMS, A. J. & SITSAPESAN, R. (2000). Evidence for Ca²⁺ activation and inactivation sites on the luminal side of the cardiac ryanodine receptor complex. Circulation Research 87, 201-206 [Abstract/Full Text]

DUKE, A. M. & STEELE, D. S. (1998a). Effects of caffeine and adenine nucleotides on Ca²⁺ release by the sarcoplasmic reticulum in saponin permeabilized frog skeletal muscle fibres. Journal of Physiology 513, 43-53 [Abstract/Full Text]

DUKE, A. M. & STEELE, D. S. (1998b). Effects of cyclopiazonic acid on Ca²⁺ regulation by the sarcoplasmic reticulum in saponin permeabilized skeletal muscle fibres. Pflügers Archiv 436, 104-111 [Medline]

DUNCAN, L., BURTON, F. L. & SMITH, G. L. (1999). REACT: Calculation of free metal and ligand concentrations using a Windows-based computer program. Journal of Physiology 517.P, 2P

ENDO, M. (1977). Calcium release from the sarcoplasmic reticulum. Physiological Reviews 57, 71-108 [Medline]

EUROPEAN MALIGNANT HYPERPYREXIA GROUP (1984). A protocol for the investigation of malignant hyperpyrexia (MH) susceptibility. British Journal of Anesthesiology 56, 1267-1269 (). FILL. M., STEFANI, E. & NELSON, T. E., 1991

FINK, R. H., STEPHENSON, D. G. & WILLIAMS, D. A. (1990). Physiological properties of skinned fibres from normal and dystrophic (Duchene) human muscle activated by Ca²⁺ and Sr²⁺. Journal of Physiology 420, 337-353 [Abstract]

FLETCHER, J. E., MAYERBERGER, S., TRIPOLITIS, L., YUDKOWSKY, M. & ROSENBERG, H. (1991). Fatty acids markedly lower the threshold for halothane-induced calcium release from the terminal cisternae in human and porcine normal and malignant hyperthermia susceptible skeletal muscle. Life Sciences 49, 1651-1657 [Medline]

FLETCHER, J. E., TRIPOLITIS, L., ROSENBERG, H. & BEECH, J. (1993). Malignant hyperthermia: halothane- and calcium-induced calcium release in skeletal muscle. Biochemistry and Molecular Biology International 29, 763-772 [Medline]

FRUEN, B. R., KANE, P. K., MICKELSON, J. R. & LOUIS, C. F. (1996). Chloride-dependent sarcoplasmic reticulum Ca²⁺ release correlates with increased Ca²⁺ activation of ryanodine receptors. Biophysical Journal 71, 2522-2530 [Abstract]

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GRYNKIEWICZ, G., POENIE, M. & TSIEN, R. Y. (1985). A new generation of Ca indicators with greatly improved fluorescence properties. Journal of Biological Chemistry 260, 3440-3450 [Abstract]

HOPKINS, P. M. (2000). Malignant hyperthermia: advances in clinical management and diagnosis. British Journal of Anaesthesiology 85, 118-128

ILES, D. E, LEHMANN-HORN, F., SCHERER, S. W., TSUI, L. C., OLDE WEGHUIS, D., SUIJKERBUIJK, R. F., HEYTENS, L., MIKALA, G., SCHWARTZ, A., ELLIS, F. R., STEWART, A. D., DEUFEL, T. & WIERINGA, B. (1994). Localization of the gene encoding the alpha 2/delta-subunits of the L-type voltage-dependent calcium channel to chromosome 7q and analysis of the segregation of flanking markers in malignant hyperthermia susceptible families. Human Molecular Genetics 3, 969-975 [Abstract]

KABBARA, A. A. & ALLEN, D. G. (2001). The use of the indicator fluo-5N to measure sarcoplasmic reticulum calcium in single muscle fibres of the cane toad. Journal of Physiology 534, 87-97 [Abstract/Full Text]

KABBARA, A. A. & STEPHENSON, D. G. (1994). Effects of Mg²⁺ on Ca²⁺ handling by the sarcoplasmic reticulum in skinned skeletal and cardiac muscle fibres. Pflügers Archiv 428, 331-339 [Medline]

KAWANA, Y., IINO, M., HORIUTI, K., MATSUMURA, N., OHTA, T., MATSUI, K. & ENDO, M. (1992). Acceleration in calcium-induced calcium release in the biopsied muscle-fibers from patients with malignant hyperthermia. Biomedical Research 13, 287-297

LAMB, G. D., CELLINI, M. A. & STEPHENSON, D. G. (2001). Different Ca²⁺ releasing action of caffeine and depolarisation in skeletal muscle fibres of the rat. Journal of Physiology 531, 715-728 [Abstract/Full Text]

LAMB, G. D. & STEPHENSON, D. G. (1994). Effects of intracellular pH and [Mg²⁺] on excitation-contraction coupling in skeletal muscle fibres of the rat. Journal of Physiology 478, 331-339 [Abstract]

LAVER, D. R., BAYNES, T. M. & DULHUNTY, A. F. (1997). Magnesium inhibition of ryanodine-receptor calcium channels: evidence for two independent mechanisms. Journal of Membrane Biology 156, 213-229 [Medline]

LEVITT, R. C., OLCKERS, A., MEYERS, S., FLETCHER, J. E., ROSENBERG, H., ISAACS, H. & MEYERS, D. A. (1992). Evidence for the localization of a malignant hyperthermia susceptibility locus (MHS2) to human chromosome 17q. Genomics 14, 562-566 [Medline]

LOUIS, C. F., ZUALKERNAN, K., ROGHAIR, T. & MICKELSON, J. R. (1992). The effects of volatile anesthetics on calcium regulation by malignant hyperthermia susceptible sarcoplasmic reticulum. Anesthesiology 77, 114-125 [Medline]

MANNING, B. M., QUANE, K. A., ORDING, H., URWYLER, A., TEGAZZIN, V., LEHANE , M., O'HALLORAN, J., HARTUNG, E., GIBLIN, L. M., LYNCH, P. J., VAUGHAN, P., CENSIER, K., BENDIXEN, D., COMI, G., HEYTENS, L., MONSIEURS, K., FAGERLUND, T., WOLZ, W., HEFFRON, J. J., MULLER, C. R. & MCCARTHY, T. V. (1998). Identification of novel mutations in the ryanodine-receptor gene (RYR1) in malignant hyperthermia: genotype-phenotype correlation American Journal of Human Genetics 62, 599-609. , MEISSNER

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MONNIER, N., PROCACCIO, V., STIEGLITZ, P. & LUNARDI, J. (1997). Malignant-hyperthermia susceptibility is associated with a mutation of the alpha 1-subunit of the human dihydropyridine-sensitive L-type voltage-dependent calcium-channel receptor in skeletal muscle. American Journal of Human Genetics 60, 1316-1325 [Medline]

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Acknowledgements

The financial support of the Wellcome Trust is acknowledged.

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ADNET, P. J., BROMBERG, N. L., HAUDECOEUR, G., KRIVOSIC, I., ADAMANTIDIS, H., REYFORD, H. & DUPUIS, B. A. (1993). Fibre-type caffeine-sensitivities in skinned muscle fibres from humans susceptible to malignant hyperthermia. Anesthesiology 78, 168-177	[Medline]
BLAZEV, R. & LAMB, G. D. (1999). Low [ATP] and elevated [Mg²⁺] reduce depolarization-induced Ca²⁺ release in rat skinned skeletal muscle fibres. Journal of Physiology 520, 203-215	[Abstract/Full Text]
BRANDT, A., SCHLEITHOFF, L., JURKAT-ROTT, K., KLINGER, W., BAUR, C. & LEHMANN-HORN, F. (1999). Screening of the ryanodine receptor gene in 105 malignant hyperthermia families: novel mutations and concordance with the in vitro contracture test. Human Molecular Genetics 8, 2055-2062	[Abstract/Full Text]
CHING, L. L., WILLIAMS, A. J. & SITSAPESAN, R. (2000). Evidence for Ca²⁺ activation and inactivation sites on the luminal side of the cardiac ryanodine receptor complex. Circulation Research 87, 201-206	[Abstract/Full Text]
DUKE, A. M. & STEELE, D. S. (1998a). Effects of caffeine and adenine nucleotides on Ca²⁺ release by the sarcoplasmic reticulum in saponin permeabilized frog skeletal muscle fibres. Journal of Physiology 513, 43-53	[Abstract/Full Text]
DUKE, A. M. & STEELE, D. S. (1998b). Effects of cyclopiazonic acid on Ca²⁺ regulation by the sarcoplasmic reticulum in saponin permeabilized skeletal muscle fibres. Pflügers Archiv 436, 104-111	[Medline]
DUNCAN, L., BURTON, F. L. & SMITH, G. L. (1999). REACT: Calculation of free metal and ligand concentrations using a Windows-based computer program. Journal of Physiology 517.P, 2P
ENDO, M. (1977). Calcium release from the sarcoplasmic reticulum. Physiological Reviews 57, 71-108	[Medline]
EUROPEAN MALIGNANT HYPERPYREXIA GROUP (1984). A protocol for the investigation of malignant hyperpyrexia (MH) susceptibility. British Journal of Anesthesiology 56, 1267-1269 (). FILL. M., STEFANI, E. & NELSON, T. E., 1991
FINK, R. H., STEPHENSON, D. G. & WILLIAMS, D. A. (1990). Physiological properties of skinned fibres from normal and dystrophic (Duchene) human muscle activated by Ca²⁺ and Sr²⁺. Journal of Physiology 420, 337-353	[Abstract]
FLETCHER, J. E., MAYERBERGER, S., TRIPOLITIS, L., YUDKOWSKY, M. & ROSENBERG, H. (1991). Fatty acids markedly lower the threshold for halothane-induced calcium release from the terminal cisternae in human and porcine normal and malignant hyperthermia susceptible skeletal muscle. Life Sciences 49, 1651-1657	[Medline]
FLETCHER, J. E., TRIPOLITIS, L., ROSENBERG, H. & BEECH, J. (1993). Malignant hyperthermia: halothane- and calcium-induced calcium release in skeletal muscle. Biochemistry and Molecular Biology International 29, 763-772	[Medline]
FRUEN, B. R., KANE, P. K., MICKELSON, J. R. & LOUIS, C. F. (1996). Chloride-dependent sarcoplasmic reticulum Ca²⁺ release correlates with increased Ca²⁺ activation of ryanodine receptors. Biophysical Journal 71, 2522-2530	[Abstract]
FRYER, M. W. & STEPHENSON, D. G. (1996). Total and sarcoplasmic reticulum calcium contents of skinned fibres from rat skeletal muscle. Journal of Physiology 493, 357-370	[Abstract]
FUJII, J., OTSU, K., ZORZATO, F., DE LEON, S., KHANNA, V. K., WEILER, J. E., O'BRIEN, P. J. & MACLENNAN, D. H. (1991). Identification of a mutation in porcine ryanodine receptor associated with malignant hyperthermia. Science 253, 448-451
GRYNKIEWICZ, G., POENIE, M. & TSIEN, R. Y. (1985). A new generation of Ca indicators with greatly improved fluorescence properties. Journal of Biological Chemistry 260, 3440-3450	[Abstract]
HOPKINS, P. M. (2000). Malignant hyperthermia: advances in clinical management and diagnosis. British Journal of Anaesthesiology 85, 118-128
ILES, D. E, LEHMANN-HORN, F., SCHERER, S. W., TSUI, L. C., OLDE WEGHUIS, D., SUIJKERBUIJK, R. F., HEYTENS, L., MIKALA, G., SCHWARTZ, A., ELLIS, F. R., STEWART, A. D., DEUFEL, T. & WIERINGA, B. (1994). Localization of the gene encoding the alpha 2/delta-subunits of the L-type voltage-dependent calcium channel to chromosome 7q and analysis of the segregation of flanking markers in malignant hyperthermia susceptible families. Human Molecular Genetics 3, 969-975	[Abstract]
KABBARA, A. A. & ALLEN, D. G. (2001). The use of the indicator fluo-5N to measure sarcoplasmic reticulum calcium in single muscle fibres of the cane toad. Journal of Physiology 534, 87-97	[Abstract/Full Text]
KABBARA, A. A. & STEPHENSON, D. G. (1994). Effects of Mg²⁺ on Ca²⁺ handling by the sarcoplasmic reticulum in skinned skeletal and cardiac muscle fibres. Pflügers Archiv 428, 331-339	[Medline]
KAWANA, Y., IINO, M., HORIUTI, K., MATSUMURA, N., OHTA, T., MATSUI, K. & ENDO, M. (1992). Acceleration in calcium-induced calcium release in the biopsied muscle-fibers from patients with malignant hyperthermia. Biomedical Research 13, 287-297
LAMB, G. D., CELLINI, M. A. & STEPHENSON, D. G. (2001). Different Ca²⁺ releasing action of caffeine and depolarisation in skeletal muscle fibres of the rat. Journal of Physiology 531, 715-728	[Abstract/Full Text]
LAMB, G. D. & STEPHENSON, D. G. (1994). Effects of intracellular pH and [Mg²⁺] on excitation-contraction coupling in skeletal muscle fibres of the rat. Journal of Physiology 478, 331-339	[Abstract]
LAVER, D. R., BAYNES, T. M. & DULHUNTY, A. F. (1997). Magnesium inhibition of ryanodine-receptor calcium channels: evidence for two independent mechanisms. Journal of Membrane Biology 156, 213-229	[Medline]
LEVITT, R. C., OLCKERS, A., MEYERS, S., FLETCHER, J. E., ROSENBERG, H., ISAACS, H. & MEYERS, D. A. (1992). Evidence for the localization of a malignant hyperthermia susceptibility locus (MHS2) to human chromosome 17q. Genomics 14, 562-566	[Medline]
LOUIS, C. F., ZUALKERNAN, K., ROGHAIR, T. & MICKELSON, J. R. (1992). The effects of volatile anesthetics on calcium regulation by malignant hyperthermia susceptible sarcoplasmic reticulum. Anesthesiology 77, 114-125	[Medline]
MANNING, B. M., QUANE, K. A., ORDING, H., URWYLER, A., TEGAZZIN, V., LEHANE , M., O'HALLORAN, J., HARTUNG, E., GIBLIN, L. M., LYNCH, P. J., VAUGHAN, P., CENSIER, K., BENDIXEN, D., COMI, G., HEYTENS, L., MONSIEURS, K., FAGERLUND, T., WOLZ, W., HEFFRON, J. J., MULLER, C. R. & MCCARTHY, T. V. (1998). Identification of novel mutations in the ryanodine-receptor gene (RYR1) in malignant hyperthermia: genotype-phenotype correlation American Journal of Human Genetics 62, 599-609. , MEISSNER
MICKELSON, J. R. & LOUIS, C. F. (1996). Malignant hyperthermia: excitation-contraction coupling, Ca²⁺ release channel, and cell Ca²⁺ regulation defects. Physiological Reviews 76, 537-592	[Medline]
NELSON, T. E. (1988). SR function in malignant hyperthermia. Cell Calcium 9, 257-265	[Medline]
MONNIER, N., PROCACCIO, V., STIEGLITZ, P. & LUNARDI, J. (1997). Malignant-hyperthermia susceptibility is associated with a mutation of the alpha 1-subunit of the human dihydropyridine-sensitive L-type voltage-dependent calcium-channel receptor in skeletal muscle. American Journal of Human Genetics 60, 1316-1325	[Medline]
OHNISHI, S. T., TAYLOR, S. & GRONERT, G. A. (1983). Calcium-induced Ca²⁺ release from sarcoplasmic reticulum of pigs susceptible to malignant hyperthermia. The effects of halothane and dantrolene. FEBS Letters 161, 103-107	[Medline]
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				A. M. Duke, P. M. Hopkins, P. J. Halsall, and D. S. Steele Mg2+ dependence of Ca2+ release from the sarcoplasmic reticulum induced by sevoflurane or halothane in skeletal muscle from humans susceptible to malignant hyperthermia Br. J. Anaesth., September 1, 2006; 97(3): 320 - 328. [Abstract] [Full Text] [PDF]