Free
Critical Care Medicine  |   October 2010
Lymphocyte-based Determination of Susceptibility to Malignant Hyperthermia: A Pilot Study in Swine
Author Affiliations & Notes
  • Saiid Bina, Ph.D.
    *
  • John Capacchione, M.D.
    *
  • Sheila Muldoon, M.D.
  • Munkhuu Bayarsaikhan, Ph.D.
  • Rolf Bunger, M.D., Ph.D.
    §
  • * Research Assistant Professor, † Professor, ‡ Postdoctoral Associate, Department of Anesthesiology, § Professor, Department of Anatomy, Physiology, and Genetics, Uniformed Services University of the Health Sciences, Bethesda, Maryland.
Article Information
Critical Care Medicine / Critical Care / Patient Safety
Critical Care Medicine   |   October 2010
Lymphocyte-based Determination of Susceptibility to Malignant Hyperthermia: A Pilot Study in Swine
Anesthesiology 10 2010, Vol.113, 917-924. doi:10.1097/ALN.0b013e3181ee2cd0
Anesthesiology 10 2010, Vol.113, 917-924. doi:10.1097/ALN.0b013e3181ee2cd0
What We Already Know about This Topic
  • ❖ Different in vitro  and genetic tests have been proposed for detection of susceptibility against malignant hyperthermia.
What This Article Tells Us That Is New
  • ❖ In lymphocytes from malignant hyperthermia-susceptible pigs, adenosine formation was greater than in normal swine.
  • ❖ Lymphocyte adenosine production in blood from normal and malignant hyperthermia-susceptible hymans should be tested to validate this new test.
MALIGNANT hyperthermia (MH) is a genetic disorder that exaggerates metabolic responses to volatile anesthetics and the depolarizing muscle relaxant succinylcholine. Exposure to these agents triggers a complex cascade of biochemical reactions and signaling that results in muscle rigidity, rhabdomyolysis, cardiac arrhythmia, and potentially lethal hyperthermia.1,2 If not treated promptly by withdrawing anesthesia and administering dantrolene, an intracellular calcium antagonist, mortality can exceed 80%. Early signs and symptoms of an imminent MH episode can be slow and clinically ambiguous, making MH difficult to recognize. Identification of affected persons is through a caffeine-halothane contracture test (CHCT) on biopsied leg vastus lateralis muscle, with slight differences in the North America CHCT3 and European in vitro  contracture test4 protocols. CHCT has a sensitivity of 97% (accurately detects those with MH) and a specificity of 78% (yields some false positives).5 Muscle fibers from MH-positive persons are markedly more sensitive to caffeine and halothane. The reproducible shift in the dose sensitivity of muscle contracture to these compounds has led to its use as the diagnostic indicator of MH.6 However, because of the invasive nature of the test, its costs, and associated risks, it is estimated that only approximately 10% of eligible patients undergo CHCT (projected from referrals to the database of the North America Malignant Hyperthermia Hotline). Thus, a functional, minimally invasive, safe, relatively economical, and simple MH diagnostic test is desirable.
Several alternative MH tests have been considered in the literature as being less or minimally invasive. The most extensively investigated approach is molecular testing of the ryanodine receptor type 1 (RyR1  ) gene.7–11 However, because the total number of mutations that could potentially alter or affect ryanodine receptor (RyR) function is not yet known, genetic screening has not been, and likely will not soon be, definitive enough to serve as the sole method for diagnosing MH. Functional MH tests using microdialysis,12–14 immortalized B cells-Ca2+assay,15,16 and nuclear magnetic resonance spectroscopy17 are either modestly invasive (microdialysis) or too technically involved and time consuming to be practical for use in MH diagnostic testing.
In the present study, we examine a novel blood test for utility in MH diagnosis using the well-characterized porcine MH model. Our approach isolates and compares lymphocytes (B + T cells) from swine carrying a single MH mutation, Arg615Cys (referred to as “MHS swine” throughout), with lymphocytes from normal swine to determine whether significant differences in adenosine levels could be measured to distinguish the two from one another. 4-Chloro-m-cresol (4CmC), a chlorinated phenol that mimics the effects of caffeine and halothane by inducing release of sarcoplasmic reticulum (SR) Ca2+in skeletal muscles,18 as well as in isolated SR vesicles19, was used to stimulate lymphocytes.
The endoplasmic reticulum/SR has two families of intracellular ion channels capable of releasing Ca2+: the inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) and the RyRs. IP3R agonists acting through G-protein-linked or tyrosine kinase-linked receptors stimulate phospholipase C to generate the second messenger IP3, which then diffuses into the cytoplasm to release stored Ca2+by binding to IP3R.20,21 IP3R can be activated by adenophostin A and inhibited by xestospongin C (XeC).22 RyRs are found in muscle cells, neurons, and other cell types.21–23 RyR-induced Ca2+release is activated by caffeine and 4CmC19 but is inhibited by dantrolene/azumolene.24 A correlation between cell Ca2+release leading to increased Ca2+ATPase-dependent Ca2+pumping and subsequent adenosine formation in lymphocytes has not been reported. However, in skeletal muscle, RyR1 is coupled to the dihydropyridine receptor. The dihydropyridine receptor is the voltage-sensitive L-type Ca2+channel. Mitochondria (the site of oxidative phosphorylation of adenosine 5′-diphosphate [ADP]) and glycolysis (the cytoplasmic site of substrate-level phosphorylation of ADP) synthesize adenosine 5′-triphosphate (ATP), which powers the ATP-dependent Ca2+pumps on the SR/endoplasmic reticulum and the sarcolemma. Intracellular Ca2+accumulation due to the activation and subsequent nonclosure/delay-closure of the RyR1 Ca2+channel triggers this ATPase-dependent Ca2+pumping, generating ADP and adenosine 5′-monophosphate (via  adenylate kinase), leading to increased adenosine formation.25–27 
We demonstrate that (1) adenosine formation by 4CmC-stimulated lymphocytes can be used to assess increased adenosine ATP turnover and catabolism resulting in increased adenosine formation; (2) defined mutation in RyR1 that leads to abnormal agonist-induced Ca2+release in skeletal muscle also leads to increase adenosine formation by lymphocytes; and (3) 4CmC-induced adenosine production is substantially greater in lymphocytes from MHS swine compared with normal swine. We attempted to identify Ca2+pools that could possibly contribute to measured 4CmC-induced adenosine formation in lymphocytes from normal swine by using specific blockers of IP3R, mitochondrial oxidative phosphorylation, and SR Ca2+release channels. We also considered extracellular Ca2+as a possible effector of intracellular Ca2+changes, thus potentially contributing to increased adenosine formation.
Materials and Methods
Experiments were designed to (1) examine the hypothesis that a defined mutation in the RyR1 gene of MHS swine that leads to abnormal agonist-induced Ca2+release in skeletal muscle will also lead to adenosine release in lymphocytes; (2) determine whether 4CmC stimulation of lymphocytes and adenosine production distinguishes between MHS and normal swine, and (3) delineate the possible effects of IP3R, mitochondrial uncoupling, external Ca2+, and the SR Ca2+release channels (RyR1) on the observed adenosine formation.
Animal Model
With approval of the Uniformed Services University of the Health Sciences (Bethesda, Maryland) institutional animal care and use committee, seven normal pigs (not MH-sensitive; Archer Farm, Darlington, MD) and seven homozygous MHS swine (Boyle Farms, Moorhead, IA) carrying an Arg615Cys mutation were sedated with ketamine (8-10 mg/kg intramuscular injection). An ear vein was cannulated for inducing and maintaining anesthesia with propofol (0.2-0.4 mg · kg−1· min−1). An endotracheal tube was inserted and ventilation was maintained with an end-tidal carbon dioxide of 40 ± 5 mmHg. A rectal thermometer probe was placed to monitor and maintain core body temperature at 38.5°C. A 20-gauge cannula was inserted percutaneously into the superficial femoral artery. Blood pressure and heart rate were monitored continuously. Efforts were made to match the two experimental animal groups in regard to factors such as sex, age, or weight. After cardiovascular and respiratory indices stabilized, an arterial blood sample (40 ml) was withdrawn using a heparinized 60-ml syringe. After blood sampling, a skeletal muscle biopsy (vastus lateralis) for CHCT was performed as described previously.3 Swine were then administered inhaled halothane (2%) for the induction of an MHS clinical episode.
Isolation of Lymphocytes from Whole Blood
Lymphocytes (B + T cells) were isolated from whole blood by the Ficoll-Hypaque density gradient centrifugation technique.28 Isolated lymphocyte cells were dispensed into 3-volume normal Hank's balanced salt solution (HBSS) and centrifuged (10 min at 1,000 revolutions per minute). The pellet was resuspended in HBSS to lyse contaminating erythrocytes and spun; this step was repeated once more. No further purification was attempted to maintain simplicity of the protocol for potential applicability in a possible MH diagnostic test in routine clinical practice. Lymphocytes were then cultured in RPMI-1640 media supplemented with 10% inactivated fetal bovine serum plus 100 units/ml penicillin and 2 mm glutamine. Cell cultures were incubated at 37°C in a humidified chamber with 5% CO2. All experiments were completed within 4 days after isolation of lymphocytes from whole blood samples.
Nucleosides Assay Protocol
Lymphocytes (1-2 × 106cells/test; cell counting was performed using hemacytometer) were suspended in HBSS with or without Ca2+/Mg2+as indicated for each experiment plus 0.1% endotoxin-free bovine serum albumin. Experiments were carried out in the presence of adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl)-adenine-HCl (EHNA; 0.1 mm) to prevent deamination of adenosine to inosine29 and incubated at 37°C for 10 min followed by treatment with different 4CmC concentrations (0.2-10 mm) and then incubated at 37°C for an additional 45 min. Final volume of the test tubes mixture were adjusted to 0.2 ml using HBSS buffer. Termination of adenosine metabolism and purification of samples were accomplished by addition of ice cold 0.1 ml 12% perchloric acid followed by centrifugation at 7,000 revolutions per minute for 10 min. Aliquots of supernatant (50 μl) were assayed for adenosine and inosine content using the high performance liquid chromatography technique detailed below. The protocols used to study the effects of external Ca2+/Mg2+, SR Ca2+release blocker azumolene, inhibitor of IP3R xestospongin C (XeC), and mitochondria uncoupler carbonyl cyanide 4-(trifluoromethoxy) phenylhydrozone (FCCP), as well as the effect of EHNA, were the same as those given in the nucleoside assay protocol. Further details and specific conditions for each experiment are presented in the result section and figure legends.
High-Performance Liquid Chromatography Analysis
A chromatography protocol was developed using a high-performance liquid chromatography system (Waters Separations module, model 2695; Waters Co., Milford, MA) consisted of a Waters Symmetry C18column (4.6 × 250 mm) and mobile phase of 94% KH2PO4(50 mm, pH 4.6) containing 1-heptanesulfonic acid (0.5 mm) and 6% acetonitrile. Flow rate was set at 1 ml/min. Under these conditions, retention times for inosine and adenosine were 3.6 and 6.6 min, respectively. Peak width of inosine and adenosine were 18 and 36 s, respectively. Eluted nucleosides were detected by their absorbance at λ= 254 nm using Waters photodiode array detector (model 996). Sample adenosine and inosine concentrations were interpolated from a calibrated standard curve of concentrations versus  peak area.
Statistics
Sample nucleoside concentrations were normalized to represent per 106cells and presented as mean ± SEM. SigmaPlot (version 10) and SigmaStat (version 3.5) software (Systat Software, Inc., Richmond, CA) were used to plot graphs and perform statistics, respectively. Pairwise comparisons using two-way analysis of variance with Bonferroni correction (α set to 0.05) were applied to test for statistical differences between multiple treatment groups. In addition, two-tailed t  tests were done comparing two treatments of interest, and exact P  values are presented in the text; P  less than 0.05 indicated statistical significance.
Reagents
HBSS containing Ca2+/Mg2+(0.4 mm Ca2+and 0.5 mm Mg2+, referred to as normal HBSS) and Ca2+/Mg2+-free HBSS were from Invitrogen/GIBCO (Carlsbad, CA). The adenosine deaminase inhibitor EHNA (Sigma-Aldrich, St. Louis, MO) was dissolved in either normal HBSS or Ca2+/Mg2+-free HBSS (Sigma-Aldrich), depending on the protocol used, and stored at −20°C. KH2PO4, 1-heptanesulfonic acid, bovine serum albumin, adenosine, inosine, and other ordinary laboratory chemicals were from Sigma-Aldrich. RPMI-1640 culture media, fetal bovine serum, and penicillin were from Quality Biologic Inc. (Gaithersburg, MD). 4CmC (Sigma-Aldrich) was prepared daily in distilled water. Carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP; Sigma-Aldrich) and XeC (Calbiochem, San Diego, CA) were dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich) and stored at −20°C. Azumolene was dissolved in DMSO and stored at −20°C.
Results
Although seven lymphocyte cell lines in each group of MHS and normal swine were examined, the numbers of cell lines in each protocol were variable due to limited availability of cells. Lymphocytes from MHS and normal swine were used to examine the effects of adenosine deaminase inhibitor EHNA on adenosine formation and on 4CmC dose-response experiments. Due to limited availability of the porcine MHS lymphocytes, only lymphocytes from normal swine were used to study the effects of external Ca2+, azumolene, XeC, and uncoupler FCCP on adenosine formation. In preliminary tests, we observed that adenosine formation in response to 4CmC was a function of the length of the cell culturing period. We found that after culturing cells for 10 days, more than 90% of the 4CmC effectiveness on lymphocytes was abolished in terms of adenosine formation (data not shown).
Effect of Adenosine Deaminase Inhibitor on Adenosine/Inosine Levels
Figure 1shows 4CmC (1 mm)-induced adenosine/inosine formation in the presence and absence of 0.1 mm EHNA. The lack or weak formation of adenosine response to 4CmC stimulation was due to immediate deamination of adenosine to inosine by the endogenous enzyme adenosine deaminase. This was confirmed when lymphocyte cells were incubated with various concentrations (0-1,000 μm) of EHNA. The results demonstrated that at EHNA concentrations of 20 μm or higher, inosine formation decreased and adenosine concentration increased to plateau level (n = 3, EHNA dose-response data not shown). The observed differences in the molar concentrations of inosine and adenosine in figure 1were likely due to degradation of inosine to hypoxanthine by endogenous purine nucleoside phosphorylase and, at least in part, also due to the differences in the molar absorption coefficient (ϵ) of inosine compared with adenosine (the measuring wavelength was λ= 254 nm; adenosine, λmax= 258 nm, ϵ= 15,100; inosine, λmax= 248 nm, ϵ= 12,200 [Merck Index]).
Fig. 1.  Effect of adenosine deaminase inhibitor erythro-9(2-hydroxy-3-nonyl) adenine (EHNA, 0.1 mm) on 1 mm 4-chloro-m-cresol (4CmC)-induced adenosine/inosine in lymphocyte cells from swine carrying the Arg615Cys mutation and having positive caffeine-halothane contracture test (CHCT). Sample nucleoside concentrations were normalized to reflect values per 106cells. Lymphocyte cells (1-2 × 106) from malignant hyperthermia-susceptible (n = 4) swine were suspended at 37°C for 10 min in normal Ca2+/Mg2+-containing Hank's balanced salt solution in the absence and presence of EHNA followed by treatment with 1 mm 4CmC and continuation of incubation for additional 30 min (for further details see Methods). *In the absence of EHNA, inosine level is significantly higher than adenosine level, but in the presence of EHNA, adenosine level is significantly higher than inosine level.
Fig. 1. 
	Effect of adenosine deaminase inhibitor erythro-9(2-hydroxy-3-nonyl) adenine (EHNA, 0.1 mm) on 1 mm 4-chloro-m-cresol (4CmC)-induced adenosine/inosine in lymphocyte cells from swine carrying the Arg615Cys mutation and having positive caffeine-halothane contracture test (CHCT). Sample nucleoside concentrations were normalized to reflect values per 106cells. Lymphocyte cells (1-2 × 106) from malignant hyperthermia-susceptible (n = 4) swine were suspended at 37°C for 10 min in normal Ca2+/Mg2+-containing Hank's balanced salt solution in the absence and presence of EHNA followed by treatment with 1 mm 4CmC and continuation of incubation for additional 30 min (for further details see Methods). *In the absence of EHNA, inosine level is significantly higher than adenosine level, but in the presence of EHNA, adenosine level is significantly higher than inosine level.
Fig. 1.  Effect of adenosine deaminase inhibitor erythro-9(2-hydroxy-3-nonyl) adenine (EHNA, 0.1 mm) on 1 mm 4-chloro-m-cresol (4CmC)-induced adenosine/inosine in lymphocyte cells from swine carrying the Arg615Cys mutation and having positive caffeine-halothane contracture test (CHCT). Sample nucleoside concentrations were normalized to reflect values per 106cells. Lymphocyte cells (1-2 × 106) from malignant hyperthermia-susceptible (n = 4) swine were suspended at 37°C for 10 min in normal Ca2+/Mg2+-containing Hank's balanced salt solution in the absence and presence of EHNA followed by treatment with 1 mm 4CmC and continuation of incubation for additional 30 min (for further details see Methods). *In the absence of EHNA, inosine level is significantly higher than adenosine level, but in the presence of EHNA, adenosine level is significantly higher than inosine level.
×
Effect of External Ca2+/Mg2+on 4CmC-induced Adenosine Formation
There were no differences between the basal resting adenosine levels of lymphocytes from normal swine (n = 6) incubated in normal Ca2+/Mg2+-containing HBSS and Ca2+/Mg2+-free HBSS (0.015 ± 0.003 and 0.013 ± 0.003 μm, P  = 0.7, respectively). However, using 1 mm 4CmC to induce adenosine in normal HBSS versus  Ca2+/Mg2+-free HBSS, we measured 0.217 ± 0.015 versus  0.163 ± 0.017 μm, P  = 0.037, respectively. This reflected a 24.9 ± 5.1% increase in adenosine formation in the presence of normal levels of extracellular Ca2+. At 2 mm 4CmC, the difference increased to 0.189 ± 0.015 in the presence of extracellular Ca2+and 0.111 ± 0.012 μm in the absence of extracellular Ca2+reflecting a 41 ± 2.5% increase in 4CmC-induced adenosine formation (P  = 0.002, fig. 2) in the presence of extracellular Ca2+/Mg2+.
Fig. 2.  Effect of external Ca2+/Mg2+on 4-chloro-m-cresol (4CmC)-induced adenosine formation in lymphocyte cells from normal swine (n = 6). Lymphocyte cells (1-2 × 106) were suspended in Hank's balanced salt solution (HBSS) containing adenosine deaminase inhibitor (erythro-9(2-hydroxy-3-nonyl) adenine, 0.1 mm) at 37°C for 10 min in the absence and presence of Ca2+/Mg2+. After a 10-min incubation period, samples were treated with 4CmC (1 and 2 mm) as indicated. *4CmC-induced adenosine levels in the absence of external Ca2+/Mg2+are significantly lower compared with corresponding values in the presence of external Ca2+/Mg2+(P  = 0.037 and 0.002 for 1 and 2 mm 4CmC responses, respectively).
Fig. 2. 
	Effect of external Ca2+/Mg2+on 4-chloro-m-cresol (4CmC)-induced adenosine formation in lymphocyte cells from normal swine (n = 6). Lymphocyte cells (1-2 × 106) were suspended in Hank's balanced salt solution (HBSS) containing adenosine deaminase inhibitor (erythro-9(2-hydroxy-3-nonyl) adenine, 0.1 mm) at 37°C for 10 min in the absence and presence of Ca2+/Mg2+. After a 10-min incubation period, samples were treated with 4CmC (1 and 2 mm) as indicated. *4CmC-induced adenosine levels in the absence of external Ca2+/Mg2+are significantly lower compared with corresponding values in the presence of external Ca2+/Mg2+(P 
	= 0.037 and 0.002 for 1 and 2 mm 4CmC responses, respectively).
Fig. 2.  Effect of external Ca2+/Mg2+on 4-chloro-m-cresol (4CmC)-induced adenosine formation in lymphocyte cells from normal swine (n = 6). Lymphocyte cells (1-2 × 106) were suspended in Hank's balanced salt solution (HBSS) containing adenosine deaminase inhibitor (erythro-9(2-hydroxy-3-nonyl) adenine, 0.1 mm) at 37°C for 10 min in the absence and presence of Ca2+/Mg2+. After a 10-min incubation period, samples were treated with 4CmC (1 and 2 mm) as indicated. *4CmC-induced adenosine levels in the absence of external Ca2+/Mg2+are significantly lower compared with corresponding values in the presence of external Ca2+/Mg2+(P  = 0.037 and 0.002 for 1 and 2 mm 4CmC responses, respectively).
×
4CmC-induced Adenosine in Normal and MHS Lymphocytes
Experiments for 4CmC dose-response relations were carried out in normal Ca2+/Mg2+-containing HBSS. Shown in figure 3are 4CmC dose-response data for lymphocytes from normal (n = 7) and MHS (n = 7) swine. Adenosine levels increased upon 4CmC stimulation dose dependently and peaked at near 1 mm 4CmC, reaching 0.397 ± 0.043 and 0.185 ± 0.017 μm in MHS versus  normal cells (P  = 0.0035), respectively. Adenosine levels at 0 and 0.2 mm 4CmC were 0.177 and 0.341 with P  values of 0.153 and 0.07, respectively. Figure 4shows that the individual adenosine levels due to 4CmC (1 mm) from MHS cells did not overlap with those from normal cells. Subtraction of the baseline adenosine values confirmed this result. The basal plus 4CmC (1 mm)-induced adenosine ranges for normal and MHS cells were 0.145-0.209 μm and 0.250-0.537 μm, respectively.
Fig. 3.  Dose dependence of 4-chloro-m-cresol (4CmC)-induced adenosine in lymphocyte cells from normal (open circle  , n = 7) and malignant hyperthermia-susceptible swine carrying the Arg615Cys mutation in the ryanodine receptor type 1 (filled circle  , n = 7). Sample adenosine concentrations were normalized to reflect value per 106cells. Cells (1-2 × 106) in suspension in normal Hank's balanced salt solution containing adenosine deaminase inhibitor (erythro-9(2-hydroxy-3-nonyl) adenine, 0.1 mm) were incubated at 37°C for 10 min followed by treatment for additional 30 min with different 4CmC concentrations as indicated. *4CmC-induced adenosine levels in presence of 0.5, 1, 2, or 5 mm are significantly higher in the MHS group than in the normal control group; P  values were 0.0035, 0.0035, 0.0017, and 0.0008, respectively.
Fig. 3. 
	Dose dependence of 4-chloro-m-cresol (4CmC)-induced adenosine in lymphocyte cells from normal (open circle 
	, n = 7) and malignant hyperthermia-susceptible swine carrying the Arg615Cys mutation in the ryanodine receptor type 1 (filled circle 
	, n = 7). Sample adenosine concentrations were normalized to reflect value per 106cells. Cells (1-2 × 106) in suspension in normal Hank's balanced salt solution containing adenosine deaminase inhibitor (erythro-9(2-hydroxy-3-nonyl) adenine, 0.1 mm) were incubated at 37°C for 10 min followed by treatment for additional 30 min with different 4CmC concentrations as indicated. *4CmC-induced adenosine levels in presence of 0.5, 1, 2, or 5 mm are significantly higher in the MHS group than in the normal control group; P 
	values were 0.0035, 0.0035, 0.0017, and 0.0008, respectively.
Fig. 3.  Dose dependence of 4-chloro-m-cresol (4CmC)-induced adenosine in lymphocyte cells from normal (open circle  , n = 7) and malignant hyperthermia-susceptible swine carrying the Arg615Cys mutation in the ryanodine receptor type 1 (filled circle  , n = 7). Sample adenosine concentrations were normalized to reflect value per 106cells. Cells (1-2 × 106) in suspension in normal Hank's balanced salt solution containing adenosine deaminase inhibitor (erythro-9(2-hydroxy-3-nonyl) adenine, 0.1 mm) were incubated at 37°C for 10 min followed by treatment for additional 30 min with different 4CmC concentrations as indicated. *4CmC-induced adenosine levels in presence of 0.5, 1, 2, or 5 mm are significantly higher in the MHS group than in the normal control group; P  values were 0.0035, 0.0035, 0.0017, and 0.0008, respectively.
×
Fig. 4.  Scatter plot of 1 mm 4-chloro-m-cresol (4CmC)-induced adenosine levels in lymphocyte cells from malignant hyperthermia susceptibility (MHS) swine carrying Arg615Cys mutation (n = 7) versus  normal swine (n = 7). Sample adenosine concentrations were normalized to reflect value per 106cells. Cells (1-2 × 106) in suspension in normal Hank's balanced salt solution containing adenosine deaminase inhibitor (erythro-9(2-hydroxy-3-nonyl) adenine, 0.1 mm) were incubated at 37°C for 10 min followed by treatment for additional 30 min with different 4CmC concentrations as indicated. Baseline adenosine levels before 4CmC treatments were not significant different between lymphocyte cells from MHS versus  normal swine.
Fig. 4. 
	Scatter plot of 1 mm 4-chloro-m-cresol (4CmC)-induced adenosine levels in lymphocyte cells from malignant hyperthermia susceptibility (MHS) swine carrying Arg615Cys mutation (n = 7) versus 
	normal swine (n = 7). Sample adenosine concentrations were normalized to reflect value per 106cells. Cells (1-2 × 106) in suspension in normal Hank's balanced salt solution containing adenosine deaminase inhibitor (erythro-9(2-hydroxy-3-nonyl) adenine, 0.1 mm) were incubated at 37°C for 10 min followed by treatment for additional 30 min with different 4CmC concentrations as indicated. Baseline adenosine levels before 4CmC treatments were not significant different between lymphocyte cells from MHS versus 
	normal swine.
Fig. 4.  Scatter plot of 1 mm 4-chloro-m-cresol (4CmC)-induced adenosine levels in lymphocyte cells from malignant hyperthermia susceptibility (MHS) swine carrying Arg615Cys mutation (n = 7) versus  normal swine (n = 7). Sample adenosine concentrations were normalized to reflect value per 106cells. Cells (1-2 × 106) in suspension in normal Hank's balanced salt solution containing adenosine deaminase inhibitor (erythro-9(2-hydroxy-3-nonyl) adenine, 0.1 mm) were incubated at 37°C for 10 min followed by treatment for additional 30 min with different 4CmC concentrations as indicated. Baseline adenosine levels before 4CmC treatments were not significant different between lymphocyte cells from MHS versus  normal swine.
×
Effect of Azumolene
Effect of the membrane-permeable SR Ca2+release blocker azumolene was examined in 4CmC-treated cells. Azumolene is a more soluble SR Ca2+release blocker than the clinically used dantrolene.24 Lymphocytes in the presence of EHNA were treated with DMSO (final DMSO concentration, 10% v/v) with and without different concentrations of azumolene (0-1,000 μm) and incubated at 37°C for 10 min followed by treatment with 1 mm 4CmC. As shown in figure 5, azumolene in concentrations up to 1 mm only marginally inhibited 4CmC-induced adenosine formation. Nonlinear curve fitting of these data suggested an apparent Ki value near 5 mm azumolene (data not shown). In an attempt to increase permeability of azumolene across the plasma membrane, we tested 30% (v/v) DMSO (n = 3) as a vehicle. Results suggested that 30% DMSO concentration was toxic to the cells, causing significant decrease in the 4CmC-induced adenosine (data not shown).
Fig. 5.  Effect of SR Ca2+release blocker azumolene (0-1,000 μm) on 1 mm 4-chloro-m-cresol (4CmC)-induced adenosine. Lymphocyte cells (1-2 × 106) from normal swine (n = 5) in suspension in Ca2+/Mg2+-free Hank's balanced salt solution plus adenosine deaminase inhibitor (0.1 mm (erythro-9(2-hydroxy-3-nonyl) adenine) were incubated at 37°C for 10min with azumolene up to concentrations of 1 mm. This step was followed by treatment with 1 mm 4CmC for an additional 30-45-min incubation at 37°C. Measured adenosine levels in the absence of azumolene plus 4CmC were depicted against x  = 0.1 mm, thus allowing logarithmic display of the entire dose-response curve. Azumolene in the highest concentration used only marginally inhibited 4CmC-induced adenosine formation.
Fig. 5. 
	Effect of SR Ca2+release blocker azumolene (0-1,000 μm) on 1 mm 4-chloro-m-cresol (4CmC)-induced adenosine. Lymphocyte cells (1-2 × 106) from normal swine (n = 5) in suspension in Ca2+/Mg2+-free Hank's balanced salt solution plus adenosine deaminase inhibitor (0.1 mm (erythro-9(2-hydroxy-3-nonyl) adenine) were incubated at 37°C for 10min with azumolene up to concentrations of 1 mm. This step was followed by treatment with 1 mm 4CmC for an additional 30-45-min incubation at 37°C. Measured adenosine levels in the absence of azumolene plus 4CmC were depicted against x 
	= 0.1 mm, thus allowing logarithmic display of the entire dose-response curve. Azumolene in the highest concentration used only marginally inhibited 4CmC-induced adenosine formation.
Fig. 5.  Effect of SR Ca2+release blocker azumolene (0-1,000 μm) on 1 mm 4-chloro-m-cresol (4CmC)-induced adenosine. Lymphocyte cells (1-2 × 106) from normal swine (n = 5) in suspension in Ca2+/Mg2+-free Hank's balanced salt solution plus adenosine deaminase inhibitor (0.1 mm (erythro-9(2-hydroxy-3-nonyl) adenine) were incubated at 37°C for 10min with azumolene up to concentrations of 1 mm. This step was followed by treatment with 1 mm 4CmC for an additional 30-45-min incubation at 37°C. Measured adenosine levels in the absence of azumolene plus 4CmC were depicted against x  = 0.1 mm, thus allowing logarithmic display of the entire dose-response curve. Azumolene in the highest concentration used only marginally inhibited 4CmC-induced adenosine formation.
×
Effect of XeC
XeC is a potent and selective inhibitor of IP3R coupled to a special intracellular pool of Ca2.20–22,29–31 To determine whether IP3R contributed to the observed 4CmC-induced adenosine formation, normal lymphocyte cells in suspension in Ca2+/Mg2+-free HBSS were treated with DMSO (final concentration, 10% v/v) with and without XeC (1 and 10 μm). This concentration range of XeC has been reported to be an effective physiologic concentration in various cell types.20–22 Figure 6summarizes the observed effects of XeC on 4CmC-induced adenosine. XeC (1 and 10 μm) alone did not affect the basal adenosine levels (data not shown). Likewise, 1 μm XeC did not affect 4CmC-induced adenosine. However 10 μm XeC significantly decreased 4CmC-induced adenosine by 27% (P  = 0.002), suggesting that IP3R-coupled intracellular Ca2+stored in porcine lymphocytes, may contribute, in part, to 4CmC-observed adenosine formation (see fig. 3).
Fig. 6.  Effect of inositol 1,4,5-triphosphate receptor-blocker xestospongin C (XeC) on 4-chloro-m-cresol-induced adenosine formation by porcine lymphocytes cells. Cells (1-2 × 106) from normal swine (n = 5) were suspended in Ca2+/Mg2+-free Hank's balanced salt solution plus adenosine deaminase inhibitor ((erythro-9(2-hydroxy-3-nonyl) adenine, 1 mm). Samples were treated for 10 min at 37°C with different XeC concentrations as indicated in the figure. This protocol phase was followed by treatment with 2 mm 4CmC and incubation at 37°C for additional 30 min. *XeC (10 μM) significantly (P  = 0.002) reduced 4CmC (2 mm)-induced adenosine formation.
Fig. 6. 
	Effect of inositol 1,4,5-triphosphate receptor-blocker xestospongin C (XeC) on 4-chloro-m-cresol-induced adenosine formation by porcine lymphocytes cells. Cells (1-2 × 106) from normal swine (n = 5) were suspended in Ca2+/Mg2+-free Hank's balanced salt solution plus adenosine deaminase inhibitor ((erythro-9(2-hydroxy-3-nonyl) adenine, 1 mm). Samples were treated for 10 min at 37°C with different XeC concentrations as indicated in the figure. This protocol phase was followed by treatment with 2 mm 4CmC and incubation at 37°C for additional 30 min. *XeC (10 μM) significantly (P 
	= 0.002) reduced 4CmC (2 mm)-induced adenosine formation.
Fig. 6.  Effect of inositol 1,4,5-triphosphate receptor-blocker xestospongin C (XeC) on 4-chloro-m-cresol-induced adenosine formation by porcine lymphocytes cells. Cells (1-2 × 106) from normal swine (n = 5) were suspended in Ca2+/Mg2+-free Hank's balanced salt solution plus adenosine deaminase inhibitor ((erythro-9(2-hydroxy-3-nonyl) adenine, 1 mm). Samples were treated for 10 min at 37°C with different XeC concentrations as indicated in the figure. This protocol phase was followed by treatment with 2 mm 4CmC and incubation at 37°C for additional 30 min. *XeC (10 μM) significantly (P  = 0.002) reduced 4CmC (2 mm)-induced adenosine formation.
×
Effect of Uncoupler FCCP on 4CmC-induced Adenosine in Lymphocyte Cells
Using normal lymphocytes in the absence of extracellular Ca2+/Mg2+, FCCP (2 and 20 μm) alone increased basal adenosine levels several-fold (up to 350%) to 0.1-0.12 μm (fig. 7, bars A, P  < 0.008), which is consistent with ATP catabolism due to mitochondrial uncoupling. Micromolar concentrations of FCCP are known to effectively uncouple mitochondrial respiration from oxidative phosphorylation of adenosine in many cell types.32–34 Combining FCCP with subsequent 4CmC (2 mm) did not further increase adenosine formation (fig. 7, bars B), most likely reflecting Ca2+depletion of the SR due to previous FCCP treatment.
Fig. 7.  Effect of uncoupler carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) alone (bars A) and its affect on 4-chloro-m-cresol (4CmC)-induced adenosine formation (bars B) by normal swine lymphocyte cells (n = 5). In brief, one set of lymphocytes was suspended in Ca2+/Mg2+-free Hank's balanced salt solution plus adenosine deaminase inhibitor ((erythro-9(2-hydroxy-3-nonyl) adenine, 1 mm), with and without FCCP at 37°C for 10 min (bars A). In the second set of samples, cells were treated the same as in the first set; then, after the 10-min incubation period, lymphocytes were treated with 2 mm 4CmC and incubated for an additional 30 min at 37°C (bars B). Adenosine data were normalized to represent value per 106cells. *FCCP (2 and 20 μm) alone significantly increased basal adenosine levels. #In the absence of FCCP, 4CmC-induced adenosine formation was 5.8-fold higher compared with basal adenosine level.
Fig. 7. 
	Effect of uncoupler carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) alone (bars A) and its affect on 4-chloro-m-cresol (4CmC)-induced adenosine formation (bars B) by normal swine lymphocyte cells (n = 5). In brief, one set of lymphocytes was suspended in Ca2+/Mg2+-free Hank's balanced salt solution plus adenosine deaminase inhibitor ((erythro-9(2-hydroxy-3-nonyl) adenine, 1 mm), with and without FCCP at 37°C for 10 min (bars A). In the second set of samples, cells were treated the same as in the first set; then, after the 10-min incubation period, lymphocytes were treated with 2 mm 4CmC and incubated for an additional 30 min at 37°C (bars B). Adenosine data were normalized to represent value per 106cells. *FCCP (2 and 20 μm) alone significantly increased basal adenosine levels. #In the absence of FCCP, 4CmC-induced adenosine formation was 5.8-fold higher compared with basal adenosine level.
Fig. 7.  Effect of uncoupler carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) alone (bars A) and its affect on 4-chloro-m-cresol (4CmC)-induced adenosine formation (bars B) by normal swine lymphocyte cells (n = 5). In brief, one set of lymphocytes was suspended in Ca2+/Mg2+-free Hank's balanced salt solution plus adenosine deaminase inhibitor ((erythro-9(2-hydroxy-3-nonyl) adenine, 1 mm), with and without FCCP at 37°C for 10 min (bars A). In the second set of samples, cells were treated the same as in the first set; then, after the 10-min incubation period, lymphocytes were treated with 2 mm 4CmC and incubated for an additional 30 min at 37°C (bars B). Adenosine data were normalized to represent value per 106cells. *FCCP (2 and 20 μm) alone significantly increased basal adenosine levels. #In the absence of FCCP, 4CmC-induced adenosine formation was 5.8-fold higher compared with basal adenosine level.
×
CHCT Results
Halothane (3%)-induced contractures in muscle strips from MHS and normal swine were 19.2 ± 3.1 mN (range, 11.2-33.1 mN) and 1.2 ± 1.0 mN (range, 0-1.8 mN), respectively. Caffeine (2 mm)-induced contractures in muscle strips from MHS and normal swine were 10.2 ± 1.8 mN (range, 4.7-17.1 mN) and 0.4 ± 0.2 mN (range, 0-1 mN), respectively. All MHS swine developed MH episodes in response to 2% halothane, whereas the normal swine did not.
Discussion
The major findings of this pilot study may be summarized as follows: (1) 4CmC treatment of lymphocytes from MHS and normal swine produces cellular energetic stress that is indexed by increased adenosine formation; (2) 4CmC-induced adenosine formation in lymphocytes distinguishes between MHS and normal swine because there is no overlap between groups; (3) extracellular Ca2+significantly influences 4CmC-induced adenosine formation in swine lymphocytes, indicating increased transmembrane Ca2+fluxes during cellular de-energization; (4) azumolene, a SR Ca2+release blocker, in concentrations up to 1 mm, did not significantly decrease 4CmC-induced adenosine in lymphocytes from normal swine; (5) IP3R-linked Ca2+release may also contribute to 4CmC-induced adenosine; (6) uncoupled mitochondria result in near-maximal adenosine formation due to ATP depletion and possibly mitochondrial matrix Ca2+depletion; and (7) evaluation in human lymphocytes is needed to clarify whether the use of adenosine as an index of cellular Ca2+stress and pumping in lymphocytes has clinical potential as an MH diagnostic blood test.
In this study, only a relatively small number of MHS animals were examined; nevertheless, the data showed that there was no overlap between 4CmC-induced adenosine values in the MHS group versus  the normal group. However, until additional experiments are performed, it cannot be stated with certainty that by increasing number of animals in each group, overlap between 4CmC-induced adenosine will not occur. The overlap in adenosine levels may be expected to be greater in humans because there are more mutations found to be causal for MH than in the purebred swine.
There are similarities and differences between 4CmC-induced Ca2+release via  the RyR1-sensitive stores in Epstein-Barr virus immortalized human B cells15,16 and the present finding on adenosine formation by 4CmC stimulated swine lymphocytes. 4CmC and caffeine-induced Ca2+release are partially blocked by azumolene, a more water-soluble blocker of SR Ca2+release than sodium dantrolene. Using the Dakiki cell line, McKinney et al.  34 showed that 0.1 and 0.4 mm azumolene inhibited 4CmC-induced Ca2+release by 23 and 50%, respectively. However, in the present study, 1 mm azumolene, the highest concentration applied, inhibited 4CmC-induced adenosine formation in normal swine lymphocytes only by 14%, and this effect was not statistically significant. An explanation for this discrepancy is not immediately obvious but could possibly be related to different experimental conditions in our nonimmortalized swine lymphocytes compared with the Epstein-Barr virus immortalized Dakiki B cell line protocols,34 as a result of differences between cell lines from two different species and the differences in 4CmC-induced Ca2+and adenosine release mechanism.
The data suggested that removal of external Ca2+/Mg2+eliminated net Ca2+influx and/or enabled increased net Ca2+efflux across the plasma membrane in presence of 4CmC, and that such transmembrane Ca2+fluxes contributed significantly to the overall intracellular Ca2+dysregulation during cell activation by 4CmC. Thus, the adenosine formation observed under the Ca2+-free HBSS conditions likely reflected the energetic stress and associated ADP and adenosine 5′-monophosphate formation triggered by increased Ca2+ATPase activity in response to the Ca2+release from intracellular stores.25–27 Our data show that the effect of physiologic millimolar extracellular Ca2+on 4CmC-induced adenosine formation was substantial, accounting for 24.9 ± 5.1 or 41 ± 2.5% of total adenosine production in the presence of 1 or 2 mm 4CmC, respectively, under our experimental conditions.
In addition to RyR1-linked Ca2+stores, there are other physiologically important intracellular pools of Ca2+, mainly the IP3-sensitive stores20,21 and the mitochondrial matrix Ca2+pool.32–33,35 According to our findings, it seems not unlikely that the IP3-sensitive stores, especially, could contribute to cellular Ca2+dysregulation (and adenosine formation) secondary to maximal SR Ca2+release channel agonism caused by 4CmC in swine lymphocytes (fig. 3). This notion is supported by the following facts and observations: XeC, a potent and selective inhibitor of the IP3R in several cell lines,22,31 had a concentration-dependent inhibitory effect on 4CmC-induced adenosine in normal swine lymphocytes (fig. 6). At 1 μm, XeC had no measurable effect on 4CmC-induced adenosine formation, which compares well with the lack of 1 μm XeC effects on Ca2+release in the Dakiki cell line.34 However, at 10 μm, XeC inhibited 4CmC-induced adenosine significantly by 27%, providing pharmacological evidence that IP3R-mediated Ca2+release might indirectly contribute (via  additional Ca2+release secondary to 4CmC induced Ca2+release) to 4CmC-induced adenosine formation in swine lymphocytes under our conditions.
Mitochondria are the sites of oxidative phosphorylation of ADP and the generation of reactive superoxide radicals; the mitochondrial matrix is a subcellular calcium store and therefore could contribute to cytosolic Ca2+under conditions of deenergization.33,35 In addition, it has been reported that mitochondria express RyRs on their outer membrane,36 but this conclusion could not be confirmed by other investigators.37–38 FCCP is an uncoupler of oxidative phosphorylation causing severe depolarization of the inner mitochondrial membrane, which is associated with a depletion of mitochondrial matrix Ca2+.36 Application of FCCP alone on swine lymphocyte cells substantially increased basal level of adenosine in Ca2+-free incubation media. Also this effect of FCCP compares well with the previously reported FCCP-induced Ca2+accumulation in human Epstein-Barr virus immortalized B cells.34 
Taken together, these findings demonstrate increased ATP catabolism as a result of impaired cellular calcium control; they also suggest that adenosine could be a readily measurable marker for increased susceptibility to the hypermetabolic state of malignant hyperthermia and, in addition, they are consistent with 31P-NMR data by Olgin et al.  17 in patients.
In conclusion, this article presents a novel and relatively simple protocol that uses lymphocytes and employs adenosine production as a marker to distinguish between normal and MHS in a well-described swine model. The MH-specificity of the protocol is provided by the use of RyR1 agonist 4CmC. The Ca2+contributing to adenosine responses could come from several sources, including the SR Ca2+release channels, the IP3R pool, and extracellular as well as mitochondrial Ca2+. This protocol approach may be applicable in MH diagnostic and screening tests in humans, but for validation, further studies are needed.
The authors acknowledge the generous gift of azumolene from Jerry Parness, M.D. (Professor, Department of Anesthesiology, University of Pittsburgh Medical School, Pittsburgh, Pennsylvania). They thank Peter Bedocs, M.D. (Department of Anesthesiology, Uniformed Services University of the Health Sciences, Bethesda, Maryland), for supplying blood samples from normal pigs.
References
Nelson TE, Flewellen EH: Current concepts. The malignant hyperthermia syndrome. N Engl J Med 1983; 309:416–8Nelson, TE Flewellen, EH
Heffron JJ: Malignant hyperthermia: Biochemical aspects of the acute episode. Br J Anaesth 1988; 60:274–8Heffron, JJ
Larach MG: Standardization of the caffeine halothane muscle contracture test. North American Malignant Hyperthermia Group. Anesth Analg 1989; 69:511–5Larach, MG
Anonymous: A protocol for the investigation of malignant hyperthermia (MH) susceptibility. The European Malignant Hyperpyrexia Group. Br J Anaesth 1984; 56:1267–9Anonymous,
Allen GC, Larach MG, Kunselman AR: The sensitivity and specificity of the caffeine-halothane contracture test: A report from the North American Malignant Hyperthermia Registry. The North American Malignant Hyperthermia Registry of MHAUS. Anesthesiology 1998; 88:579–88Allen, GC Larach, MG Kunselman, AR
Rosenberg H, Antognini JF, Muldoon S: Testing for malignant hyperthermia. Anesthesiology 2002; 96:232–7Rosenberg, H Antognini, JF Muldoon, S
Brini M: Ryanodine receptor defects in muscle genetic diseases. Biochem Biophys Res Commun 2004; 322:1245–55Brini, M
McCarthy TV, Quane KA, Lynch PJ: Ryanodine receptor mutations in malignant hyperthermia and central core disease. Hum Mutat 2000; 15:410–7McCarthy, TV Quane, KA Lynch, PJ
Urwyler A, Deufel T, McCarthy T, West S, European Malignant Hyperthermia Group: Guidelines for molecular genetic detection of susceptibility to malignant hyperthermia. Br J Anaesth 2001; 86:283–7Urwyler, A Deufel, T McCarthy, T West, S European Malignant Hyperthermia Group,
Girard T, Urwyler A, Censier K, Mueller CR, Zorzato F, Treves S: Genotype-phenotype comparison of the Swiss malignant hyperthermia population. Hum Mutat 2001; 18:357–8Girard, T Urwyler, A Censier, K Mueller, CR Zorzato, F Treves, S
Sei Y, Sambuughin N, Muldoon S: Malignant hyperthermia genetic testing in North America Working Group Meeting. Bethesda, Maryland. September 4-5, 2002. Anesthesiology 2004; 100:464–5Sei, Y Sambuughin, N Muldoon, S
Bina S, Cowan G, Karaian J, Muldoon S, Mongan P, Bünger R: Effects of caffeine, halothane, and 4-chloro-m-cresol on skeletal muscle lactate and pyruvate in malignant hyperthermia-susceptible and normal swine as assessed by microdialysis. Anesthesiology 2006; 104:90–100Bina, S Cowan, G Karaian, J Muldoon, S Mongan, P Bünger, R
Bina S, Muldoon S, Bünger R: Effects of ryanodine on skeletal muscle lactate and pyruvate in malignant hyperthermia-susceptible and normal swine as assessed by microdialysis. Eur J Anaesthesiol 2008; 25:48–57Bina, S Muldoon, S Bünger, R
Anetseder M, Hager M, Müller CR, Roewer N: Diagnosis of susceptibility to malignant hyperthermia by use of a metabolic test. Lancet 2002; 359:1579–80Anetseder, M Hager, M Müller, CR Roewer, N
Sei Y, Brandom BW, Bina S, Hosoi E, Gallagher KL, Wyre HW, Pudimat PA, Holman SJ, Venzon DJ, Daly JW, Muldoon S: Patients with malignant hyperthermia demonstrate an altered calcium control mechanism in B lymphocytes. Anesthesiology 2002; 97:1052–8Sei, Y Brandom, BW Bina, S Hosoi, E Gallagher, KL Wyre, HW Pudimat, PA Holman, SJ Venzon, DJ Daly, JW Muldoon, S
Girard T, Cavagna D, Padovan E, Spagnoli G, Urwyler A, Zorzato F, Treves S: B-lymphocytes from malignant hyperthermia-susceptible patients have an increased sensitivity to skeletal muscle ryanodine receptor activators. J Biol Chem 2001; 276:48077–82Girard, T Cavagna, D Padovan, E Spagnoli, G Urwyler, A Zorzato, F Treves, S
Olgin J, Argov Z, Rosenberg H, Tuchler M, Chance B: Non-invasive evaluation of malignant hyperthermia susceptibility with phosphorus nuclear magnetic resonance spectroscopy. Anesthesiology 1988; 68:507–13Olgin, J Argov, Z Rosenberg, H Tuchler, M Chance, B
Baur CP, Bellon L, Felleiter P, Fiege M, Fricker R, Glahn K, Heffron JJ, Herrmann-Frank A, Jurkat-Rott K, Klingler W, Lehane M, Ording H, Tegazzin V, Wappler F, Georgieff M, Lehmann-Horn F: A multicenter study of 4-chloro-m-cresol for diagnosing malignant hyperthermia susceptibility. Anesth Analg 2000; 90:200–5Baur, CP Bellon, L Felleiter, P Fiege, M Fricker, R Glahn, K Heffron, JJ Herrmann-Frank, A Jurkat-Rott, K Klingler, W Lehane, M Ording, H Tegazzin, V Wappler, F Georgieff, M Lehmann-Horn, F
Choisy S, Huchet-Cadiou C, Leoty C: Sarcoplasmic reticulum Ca2+release by 4-chloro-m-cresol in intact and chemically skinned ferret cardiac ventricular fibers. J Pharmacol Exp Ther 1999; 290:578–86Choisy, S Huchet-Cadiou, C Leoty, C
Berridge MJ: Inositol trisphosphate and calcium signalling. Nature 1993; 361:315–25Berridge, MJ
MacKrill JJ: Protein-protein interactions in intracellular Ca2+-release channel function. Biochem J 1999; 337:345–61MacKrill, JJ
Miyamoto S, Izumi M, Hori M, Kobayashi M, Ozaki H, Karaki H: Xestospongin C, a selective and membrane-permeable inhibitor of IP(3) receptor, attenuates the positive inotropic effect of alpha-adrenergic stimulation in guinea-pig papillary muscle. Br J Pharmacol 2000; 130:650–4Miyamoto, S Izumi, M Hori, M Kobayashi, M Ozaki, H Karaki, H
McPherson PS, Campbell KP: The ryanodine receptor/Ca2+release channel. J Biol Chem 1993; 268:13765–8McPherson, PS Campbell, KP
el-Hayek R, Parness J, Valdivia HH, Coronado R, Hogan K: Dantrolene and azumolene inhibit [3H]PN200-110 binding to porcine skeletal muscle dihydropyridine receptors. Biochem Biophys Res Commun 1992; 187:894–900el-Hayek, R Parness, J Valdivia, HH Coronado, R Hogan, K
Pessah IN, Molinski TF, Meloy TD, Wong P, Buck ED, Allen PD, Mohr FC, Mack MM: Bastadins relate ryanodine-sensitive and -insensitive Ca2+efflux pathways in skeletal SR and BC3H1 cells. Am J Physiol 1997; 272:C601–14Pessah, IN Molinski, TF Meloy, TD Wong, P Buck, ED Allen, PD Mohr, FC Mack, MM
Bünger R, Soboll S: Cytosolic adenylates and adenosine release in perfused working heart. Comparison of whole tissue with cytosolic non-aqueous fractionation analyses. Eur J Biochem 1986; 159:203–13Bünger, R Soboll, S
Schulze K, Duschek C, Lasley RD, Bünger R: Adenosine enhances cytosolic phosphorylation potential and ventricular contractility in stunned guinea pig heart: Receptor-mediated and metabolic protection. J Appl Physiol 2007; 102:1202–13Schulze, K Duschek, C Lasley, RD Bünger, R
Böyum A: Isolation of mononuclear cells and granulocytes from human blood. Isolation of monuclear cells by one centrifugation, and of granulocytes by combining centrifugation and sedimentation at 1 g. Scand J Clin Lab Invest Suppl 1968; 97:77–89Böyum, A
Lorbar M, Fenton RA, Duffy AJ, Graybill CA, Dobson JG Jr: Effect of aging on myocardial adenosine production, adenosine uptake and adenosine kinase activity in rats. J Mol Cell Cardiol 1999; 31:401–12Lorbar, M Fenton, RA Duffy, AJ Graybill, CA Dobson, JG
Ibarra C, Estrada M, Carrasco L, Chiong M, Liberona JL, Cardenas C, Díaz-Araya G, Jaimovich E, Lavandero S: Insulin-like growth factor-1 induces an inositol 1,4,5-trisphosphate-dependent increase in nuclear and cytosolic calcium in cultured rat cardiac myocytes. J Biol Chem 2004; 279:7554–65Ibarra, C Estrada, M Carrasco, L Chiong, M Liberona, JL Cardenas, C Díaz-Araya, G Jaimovich, E Lavandero, S
De Smet P, Parys JB, Callewaert G, Weidema AF, Hill E, De Smedt H, Erneux C, Sorrentino V, Missiaen L: Xestospongin C is an equally potent inhibitor of the inositol 1,4,5-trisphosphate receptor and the endoplasmic-reticulum Ca(2+) pumps. Cell Calcium 1999; 26:9–13De Smet, P Parys, JB Callewaert, G Weidema, AF Hill, E De Smedt, H Erneux, C Sorrentino, V Missiaen, L
Hajnóczky G, Csordás G, Yi M: Old players in a new role: Mitochondria-associated membranes, VDAC, and ryanodine receptors as contributors to calcium signal propagation from endoplasmic reticulum to the mitochondria. Cell Calcium 2002; 32:363–77Hajnóczky, G Csordás, G Yi, M
Buckler KJ, Vaughan-Jones RD: Effects of mitochondrial uncouplers on intracellular calcium, pH and membrane potential in rat carotid body type I cells. J Physiol 1998; 513:819–33Buckler, KJ Vaughan-Jones, RD
McKinney LC, Butler T, Mullen SP, Klein MG: Characterization of ryanodine receptor-mediated calcium release in human B cells: Relevance to diagnostic testing for malignant hyperthermia. Anesthesiology 2006; 104:1191–201McKinney, LC Butler, T Mullen, SP Klein, MG
Walsh C, Barrow S, Voronina S, Chvanov M, Petersen OH, Tepikin A: Modulation of calcium signalling by mitochondria. Biochim Biophys Acta 2009; 1787:1374–82Walsh, C Barrow, S Voronina, S Chvanov, M Petersen, OH Tepikin, A
Beutner G, Sharma VK, Giovannucci DR, Yule DI, Sheu SS: Identification of a ryanodine receptor in rat heart mitochondria. J Biol Chem 2001; 276:21482–8Beutner, G Sharma, VK Giovannucci, DR Yule, DI Sheu, SS
Salnikov V, Lukyanenko YO, Lederer WJ, Lukyanenko V: Distribution of ryanodine receptors in rat ventricular myocytes. J Muscle Res Cell Motil 2009; 30:161–70Salnikov, V Lukyanenko, YO Lederer, WJ Lukyanenko, V
Lukyanenko V, Ziman A, Lukyanenko A, Salnikov V, Lederer WJ: Functional groups of ryanodine receptors in rat ventricular cells. J Physiol 2007; 583:251–69Lukyanenko, V Ziman, A Lukyanenko, A Salnikov, V Lederer, WJ
Fig. 1.  Effect of adenosine deaminase inhibitor erythro-9(2-hydroxy-3-nonyl) adenine (EHNA, 0.1 mm) on 1 mm 4-chloro-m-cresol (4CmC)-induced adenosine/inosine in lymphocyte cells from swine carrying the Arg615Cys mutation and having positive caffeine-halothane contracture test (CHCT). Sample nucleoside concentrations were normalized to reflect values per 106cells. Lymphocyte cells (1-2 × 106) from malignant hyperthermia-susceptible (n = 4) swine were suspended at 37°C for 10 min in normal Ca2+/Mg2+-containing Hank's balanced salt solution in the absence and presence of EHNA followed by treatment with 1 mm 4CmC and continuation of incubation for additional 30 min (for further details see Methods). *In the absence of EHNA, inosine level is significantly higher than adenosine level, but in the presence of EHNA, adenosine level is significantly higher than inosine level.
Fig. 1. 
	Effect of adenosine deaminase inhibitor erythro-9(2-hydroxy-3-nonyl) adenine (EHNA, 0.1 mm) on 1 mm 4-chloro-m-cresol (4CmC)-induced adenosine/inosine in lymphocyte cells from swine carrying the Arg615Cys mutation and having positive caffeine-halothane contracture test (CHCT). Sample nucleoside concentrations were normalized to reflect values per 106cells. Lymphocyte cells (1-2 × 106) from malignant hyperthermia-susceptible (n = 4) swine were suspended at 37°C for 10 min in normal Ca2+/Mg2+-containing Hank's balanced salt solution in the absence and presence of EHNA followed by treatment with 1 mm 4CmC and continuation of incubation for additional 30 min (for further details see Methods). *In the absence of EHNA, inosine level is significantly higher than adenosine level, but in the presence of EHNA, adenosine level is significantly higher than inosine level.
Fig. 1.  Effect of adenosine deaminase inhibitor erythro-9(2-hydroxy-3-nonyl) adenine (EHNA, 0.1 mm) on 1 mm 4-chloro-m-cresol (4CmC)-induced adenosine/inosine in lymphocyte cells from swine carrying the Arg615Cys mutation and having positive caffeine-halothane contracture test (CHCT). Sample nucleoside concentrations were normalized to reflect values per 106cells. Lymphocyte cells (1-2 × 106) from malignant hyperthermia-susceptible (n = 4) swine were suspended at 37°C for 10 min in normal Ca2+/Mg2+-containing Hank's balanced salt solution in the absence and presence of EHNA followed by treatment with 1 mm 4CmC and continuation of incubation for additional 30 min (for further details see Methods). *In the absence of EHNA, inosine level is significantly higher than adenosine level, but in the presence of EHNA, adenosine level is significantly higher than inosine level.
×
Fig. 2.  Effect of external Ca2+/Mg2+on 4-chloro-m-cresol (4CmC)-induced adenosine formation in lymphocyte cells from normal swine (n = 6). Lymphocyte cells (1-2 × 106) were suspended in Hank's balanced salt solution (HBSS) containing adenosine deaminase inhibitor (erythro-9(2-hydroxy-3-nonyl) adenine, 0.1 mm) at 37°C for 10 min in the absence and presence of Ca2+/Mg2+. After a 10-min incubation period, samples were treated with 4CmC (1 and 2 mm) as indicated. *4CmC-induced adenosine levels in the absence of external Ca2+/Mg2+are significantly lower compared with corresponding values in the presence of external Ca2+/Mg2+(P  = 0.037 and 0.002 for 1 and 2 mm 4CmC responses, respectively).
Fig. 2. 
	Effect of external Ca2+/Mg2+on 4-chloro-m-cresol (4CmC)-induced adenosine formation in lymphocyte cells from normal swine (n = 6). Lymphocyte cells (1-2 × 106) were suspended in Hank's balanced salt solution (HBSS) containing adenosine deaminase inhibitor (erythro-9(2-hydroxy-3-nonyl) adenine, 0.1 mm) at 37°C for 10 min in the absence and presence of Ca2+/Mg2+. After a 10-min incubation period, samples were treated with 4CmC (1 and 2 mm) as indicated. *4CmC-induced adenosine levels in the absence of external Ca2+/Mg2+are significantly lower compared with corresponding values in the presence of external Ca2+/Mg2+(P 
	= 0.037 and 0.002 for 1 and 2 mm 4CmC responses, respectively).
Fig. 2.  Effect of external Ca2+/Mg2+on 4-chloro-m-cresol (4CmC)-induced adenosine formation in lymphocyte cells from normal swine (n = 6). Lymphocyte cells (1-2 × 106) were suspended in Hank's balanced salt solution (HBSS) containing adenosine deaminase inhibitor (erythro-9(2-hydroxy-3-nonyl) adenine, 0.1 mm) at 37°C for 10 min in the absence and presence of Ca2+/Mg2+. After a 10-min incubation period, samples were treated with 4CmC (1 and 2 mm) as indicated. *4CmC-induced adenosine levels in the absence of external Ca2+/Mg2+are significantly lower compared with corresponding values in the presence of external Ca2+/Mg2+(P  = 0.037 and 0.002 for 1 and 2 mm 4CmC responses, respectively).
×
Fig. 3.  Dose dependence of 4-chloro-m-cresol (4CmC)-induced adenosine in lymphocyte cells from normal (open circle  , n = 7) and malignant hyperthermia-susceptible swine carrying the Arg615Cys mutation in the ryanodine receptor type 1 (filled circle  , n = 7). Sample adenosine concentrations were normalized to reflect value per 106cells. Cells (1-2 × 106) in suspension in normal Hank's balanced salt solution containing adenosine deaminase inhibitor (erythro-9(2-hydroxy-3-nonyl) adenine, 0.1 mm) were incubated at 37°C for 10 min followed by treatment for additional 30 min with different 4CmC concentrations as indicated. *4CmC-induced adenosine levels in presence of 0.5, 1, 2, or 5 mm are significantly higher in the MHS group than in the normal control group; P  values were 0.0035, 0.0035, 0.0017, and 0.0008, respectively.
Fig. 3. 
	Dose dependence of 4-chloro-m-cresol (4CmC)-induced adenosine in lymphocyte cells from normal (open circle 
	, n = 7) and malignant hyperthermia-susceptible swine carrying the Arg615Cys mutation in the ryanodine receptor type 1 (filled circle 
	, n = 7). Sample adenosine concentrations were normalized to reflect value per 106cells. Cells (1-2 × 106) in suspension in normal Hank's balanced salt solution containing adenosine deaminase inhibitor (erythro-9(2-hydroxy-3-nonyl) adenine, 0.1 mm) were incubated at 37°C for 10 min followed by treatment for additional 30 min with different 4CmC concentrations as indicated. *4CmC-induced adenosine levels in presence of 0.5, 1, 2, or 5 mm are significantly higher in the MHS group than in the normal control group; P 
	values were 0.0035, 0.0035, 0.0017, and 0.0008, respectively.
Fig. 3.  Dose dependence of 4-chloro-m-cresol (4CmC)-induced adenosine in lymphocyte cells from normal (open circle  , n = 7) and malignant hyperthermia-susceptible swine carrying the Arg615Cys mutation in the ryanodine receptor type 1 (filled circle  , n = 7). Sample adenosine concentrations were normalized to reflect value per 106cells. Cells (1-2 × 106) in suspension in normal Hank's balanced salt solution containing adenosine deaminase inhibitor (erythro-9(2-hydroxy-3-nonyl) adenine, 0.1 mm) were incubated at 37°C for 10 min followed by treatment for additional 30 min with different 4CmC concentrations as indicated. *4CmC-induced adenosine levels in presence of 0.5, 1, 2, or 5 mm are significantly higher in the MHS group than in the normal control group; P  values were 0.0035, 0.0035, 0.0017, and 0.0008, respectively.
×
Fig. 4.  Scatter plot of 1 mm 4-chloro-m-cresol (4CmC)-induced adenosine levels in lymphocyte cells from malignant hyperthermia susceptibility (MHS) swine carrying Arg615Cys mutation (n = 7) versus  normal swine (n = 7). Sample adenosine concentrations were normalized to reflect value per 106cells. Cells (1-2 × 106) in suspension in normal Hank's balanced salt solution containing adenosine deaminase inhibitor (erythro-9(2-hydroxy-3-nonyl) adenine, 0.1 mm) were incubated at 37°C for 10 min followed by treatment for additional 30 min with different 4CmC concentrations as indicated. Baseline adenosine levels before 4CmC treatments were not significant different between lymphocyte cells from MHS versus  normal swine.
Fig. 4. 
	Scatter plot of 1 mm 4-chloro-m-cresol (4CmC)-induced adenosine levels in lymphocyte cells from malignant hyperthermia susceptibility (MHS) swine carrying Arg615Cys mutation (n = 7) versus 
	normal swine (n = 7). Sample adenosine concentrations were normalized to reflect value per 106cells. Cells (1-2 × 106) in suspension in normal Hank's balanced salt solution containing adenosine deaminase inhibitor (erythro-9(2-hydroxy-3-nonyl) adenine, 0.1 mm) were incubated at 37°C for 10 min followed by treatment for additional 30 min with different 4CmC concentrations as indicated. Baseline adenosine levels before 4CmC treatments were not significant different between lymphocyte cells from MHS versus 
	normal swine.
Fig. 4.  Scatter plot of 1 mm 4-chloro-m-cresol (4CmC)-induced adenosine levels in lymphocyte cells from malignant hyperthermia susceptibility (MHS) swine carrying Arg615Cys mutation (n = 7) versus  normal swine (n = 7). Sample adenosine concentrations were normalized to reflect value per 106cells. Cells (1-2 × 106) in suspension in normal Hank's balanced salt solution containing adenosine deaminase inhibitor (erythro-9(2-hydroxy-3-nonyl) adenine, 0.1 mm) were incubated at 37°C for 10 min followed by treatment for additional 30 min with different 4CmC concentrations as indicated. Baseline adenosine levels before 4CmC treatments were not significant different between lymphocyte cells from MHS versus  normal swine.
×
Fig. 5.  Effect of SR Ca2+release blocker azumolene (0-1,000 μm) on 1 mm 4-chloro-m-cresol (4CmC)-induced adenosine. Lymphocyte cells (1-2 × 106) from normal swine (n = 5) in suspension in Ca2+/Mg2+-free Hank's balanced salt solution plus adenosine deaminase inhibitor (0.1 mm (erythro-9(2-hydroxy-3-nonyl) adenine) were incubated at 37°C for 10min with azumolene up to concentrations of 1 mm. This step was followed by treatment with 1 mm 4CmC for an additional 30-45-min incubation at 37°C. Measured adenosine levels in the absence of azumolene plus 4CmC were depicted against x  = 0.1 mm, thus allowing logarithmic display of the entire dose-response curve. Azumolene in the highest concentration used only marginally inhibited 4CmC-induced adenosine formation.
Fig. 5. 
	Effect of SR Ca2+release blocker azumolene (0-1,000 μm) on 1 mm 4-chloro-m-cresol (4CmC)-induced adenosine. Lymphocyte cells (1-2 × 106) from normal swine (n = 5) in suspension in Ca2+/Mg2+-free Hank's balanced salt solution plus adenosine deaminase inhibitor (0.1 mm (erythro-9(2-hydroxy-3-nonyl) adenine) were incubated at 37°C for 10min with azumolene up to concentrations of 1 mm. This step was followed by treatment with 1 mm 4CmC for an additional 30-45-min incubation at 37°C. Measured adenosine levels in the absence of azumolene plus 4CmC were depicted against x 
	= 0.1 mm, thus allowing logarithmic display of the entire dose-response curve. Azumolene in the highest concentration used only marginally inhibited 4CmC-induced adenosine formation.
Fig. 5.  Effect of SR Ca2+release blocker azumolene (0-1,000 μm) on 1 mm 4-chloro-m-cresol (4CmC)-induced adenosine. Lymphocyte cells (1-2 × 106) from normal swine (n = 5) in suspension in Ca2+/Mg2+-free Hank's balanced salt solution plus adenosine deaminase inhibitor (0.1 mm (erythro-9(2-hydroxy-3-nonyl) adenine) were incubated at 37°C for 10min with azumolene up to concentrations of 1 mm. This step was followed by treatment with 1 mm 4CmC for an additional 30-45-min incubation at 37°C. Measured adenosine levels in the absence of azumolene plus 4CmC were depicted against x  = 0.1 mm, thus allowing logarithmic display of the entire dose-response curve. Azumolene in the highest concentration used only marginally inhibited 4CmC-induced adenosine formation.
×
Fig. 6.  Effect of inositol 1,4,5-triphosphate receptor-blocker xestospongin C (XeC) on 4-chloro-m-cresol-induced adenosine formation by porcine lymphocytes cells. Cells (1-2 × 106) from normal swine (n = 5) were suspended in Ca2+/Mg2+-free Hank's balanced salt solution plus adenosine deaminase inhibitor ((erythro-9(2-hydroxy-3-nonyl) adenine, 1 mm). Samples were treated for 10 min at 37°C with different XeC concentrations as indicated in the figure. This protocol phase was followed by treatment with 2 mm 4CmC and incubation at 37°C for additional 30 min. *XeC (10 μM) significantly (P  = 0.002) reduced 4CmC (2 mm)-induced adenosine formation.
Fig. 6. 
	Effect of inositol 1,4,5-triphosphate receptor-blocker xestospongin C (XeC) on 4-chloro-m-cresol-induced adenosine formation by porcine lymphocytes cells. Cells (1-2 × 106) from normal swine (n = 5) were suspended in Ca2+/Mg2+-free Hank's balanced salt solution plus adenosine deaminase inhibitor ((erythro-9(2-hydroxy-3-nonyl) adenine, 1 mm). Samples were treated for 10 min at 37°C with different XeC concentrations as indicated in the figure. This protocol phase was followed by treatment with 2 mm 4CmC and incubation at 37°C for additional 30 min. *XeC (10 μM) significantly (P 
	= 0.002) reduced 4CmC (2 mm)-induced adenosine formation.
Fig. 6.  Effect of inositol 1,4,5-triphosphate receptor-blocker xestospongin C (XeC) on 4-chloro-m-cresol-induced adenosine formation by porcine lymphocytes cells. Cells (1-2 × 106) from normal swine (n = 5) were suspended in Ca2+/Mg2+-free Hank's balanced salt solution plus adenosine deaminase inhibitor ((erythro-9(2-hydroxy-3-nonyl) adenine, 1 mm). Samples were treated for 10 min at 37°C with different XeC concentrations as indicated in the figure. This protocol phase was followed by treatment with 2 mm 4CmC and incubation at 37°C for additional 30 min. *XeC (10 μM) significantly (P  = 0.002) reduced 4CmC (2 mm)-induced adenosine formation.
×
Fig. 7.  Effect of uncoupler carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) alone (bars A) and its affect on 4-chloro-m-cresol (4CmC)-induced adenosine formation (bars B) by normal swine lymphocyte cells (n = 5). In brief, one set of lymphocytes was suspended in Ca2+/Mg2+-free Hank's balanced salt solution plus adenosine deaminase inhibitor ((erythro-9(2-hydroxy-3-nonyl) adenine, 1 mm), with and without FCCP at 37°C for 10 min (bars A). In the second set of samples, cells were treated the same as in the first set; then, after the 10-min incubation period, lymphocytes were treated with 2 mm 4CmC and incubated for an additional 30 min at 37°C (bars B). Adenosine data were normalized to represent value per 106cells. *FCCP (2 and 20 μm) alone significantly increased basal adenosine levels. #In the absence of FCCP, 4CmC-induced adenosine formation was 5.8-fold higher compared with basal adenosine level.
Fig. 7. 
	Effect of uncoupler carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) alone (bars A) and its affect on 4-chloro-m-cresol (4CmC)-induced adenosine formation (bars B) by normal swine lymphocyte cells (n = 5). In brief, one set of lymphocytes was suspended in Ca2+/Mg2+-free Hank's balanced salt solution plus adenosine deaminase inhibitor ((erythro-9(2-hydroxy-3-nonyl) adenine, 1 mm), with and without FCCP at 37°C for 10 min (bars A). In the second set of samples, cells were treated the same as in the first set; then, after the 10-min incubation period, lymphocytes were treated with 2 mm 4CmC and incubated for an additional 30 min at 37°C (bars B). Adenosine data were normalized to represent value per 106cells. *FCCP (2 and 20 μm) alone significantly increased basal adenosine levels. #In the absence of FCCP, 4CmC-induced adenosine formation was 5.8-fold higher compared with basal adenosine level.
Fig. 7.  Effect of uncoupler carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) alone (bars A) and its affect on 4-chloro-m-cresol (4CmC)-induced adenosine formation (bars B) by normal swine lymphocyte cells (n = 5). In brief, one set of lymphocytes was suspended in Ca2+/Mg2+-free Hank's balanced salt solution plus adenosine deaminase inhibitor ((erythro-9(2-hydroxy-3-nonyl) adenine, 1 mm), with and without FCCP at 37°C for 10 min (bars A). In the second set of samples, cells were treated the same as in the first set; then, after the 10-min incubation period, lymphocytes were treated with 2 mm 4CmC and incubated for an additional 30 min at 37°C (bars B). Adenosine data were normalized to represent value per 106cells. *FCCP (2 and 20 μm) alone significantly increased basal adenosine levels. #In the absence of FCCP, 4CmC-induced adenosine formation was 5.8-fold higher compared with basal adenosine level.
×