Altered Ca2+ homeostasis in skeletal muscle is a key molecular event triggering malignant hyperthermia (MH) in malignant hyperthermia-susceptible (MHS) individuals. Genetic studies have shown that mutations in the type 1 ryanodine receptor (RYR1) are associated with MH susceptibility. Because human B lymphocytes express the RYR1, it is hypothesized that Ca2+ homeostasis in B lymphocytes is altered in MHS individuals.
This study investigated the Ca2+ response of B cells to caffeine and 4-chloro-m-cresol in 13 MHS and 21 MH-negative (MHN) individuals who had been diagnosed by caffeine halothane contracture test (CHCT) and 18 healthy volunteers. Changes in [Ca2+]i in B cells were measured directly in fluo-3 loaded cells using a dual-color flow cytometric technique. Further, B cell phenotype was correlated with CHCT results in a family with the Val2168Met (G6502A) mutation.
Caffeine-induced (50 mm) increases in [Ca2+]i in B cells were significantly greater in MHS than in MHN (P = 0.0004), control (P = 0.0001) or non-MHS (MHN and control) individuals (P < 0.0001). The 4-chloro-m-cresol-induced (400 microm) increases in [Ca2+]i were also significantly different between MHS and controls (P = 0.003) or between MHS and non-MHS (MHN and control) individuals (P = 0.0078). A study of a family with the Val2168Met mutation demonstrated expression of the RYR1 mRNA mutant in B cells from the family members with MHS phenotype and a clear segregation of genotype with B-cell phenotype.
The Ca2+ responses to caffeine or 4-chloro-m-cresol in B lymphocytes showed significant differences between MHS and MHN (or control) individuals. Although the molecular mechanisms of these alterations are currently undetermined, the results suggest that the enhanced Ca2+ responses are associated with mutations in the RYR1 gene in some MHS individuals.
MALIGNANT hyperthermia (MH) is a pharmacogenetic disorder of skeletal muscle. In MH-susceptible (MHS) individuals, halogenated anesthetic agents (halothane, enflurane, isoflurane, desflurane, and sevoflurane) and/or the depolarizing muscle relaxant, succinylcholine, trigger an abnormal acceleration of muscle metabolism that can cause a life-threatening reaction. 1–4Initial pharmacologic and biochemical studies suggest that the underlying mechanisms in MH are conditional defects in mechanisms controlling myoplasmic concentrations of [Ca2+]i. 5Consistent with an involvement of Ca2+, genetic linkage studies have shown linkage between MH and the type 1 ryanodine receptor (RYR1 ) gene of skeletal muscle on chromosome 19q13.1–13.2. 6,7To date, more than 22 mutations of RYR1 have been reported to be associated with 25–50% of MHS individuals. 7–10Further support comes from gene expression studies, which demonstrated that cells expressing mutants of RYR1 (for the 15 mutations) are more sensitive to halothane and caffeine, which initiate an increase in [Ca2+]i, than are the wild-type transfected cells. 11
Recently, we found that human B lymphocytes express not only the inositol 1,4,5-trisphosphate receptor, but also the ryanodine receptor. 12Restriction fragment length polymorphism studies, cloning, and complementary DNA (cDNA) sequencing analysis indicated that the ryanodine receptor expressed in B cells is identical to skeletal muscle type, RYR1 . 12Immunoblotting and ryanodine binding studies showed that an intact 565 kd protein is indeed expressed in B cells. The RYR-stimulating agent 4-chloro-m-cresol (4CmC) induced Ca2+release in a dose-dependent and ryanodine-sensitive fashion. Furthermore, depleting Ca2+in the ryanodine-sensitive store by 4CmC decreased the magnitude of B-cell receptor (BCR)-mediated Ca2+release, suggesting that RYR1 is involved in regulating BCR-mediated Ca2+signaling, perhaps in conjunction with the IP3receptor. 12These results suggest that RYR1 in B cells functions as a Ca2+release channel during B-cell receptor-stimulated activation. We hypothesized that Ca2+homeostasis in B cells was altered in MHS individuals. Therefore, this study compares Ca2+response induced by two RYR-stimulating agents, caffeine or 4CmC, in B cells from MHS individuals with that in B cells from MH-negative (MHN) individuals and healthy controls.
Materials and Methods
Using informed consent procedures approved by Uniformed Services University of the Health Sciences, we enrolled a total of 34 unrelated patients, who were referred to the Department for evaluation of MH status. We also enrolled five family members of a patient with the Val2168Met mutation. Blood samples were also obtained from 18 healthy volunteers. The 34 patients were referred for evaluation and diagnostic biopsy because of a personal or family history of MH or adverse metabolic response during surgery that was highly suspicious for MH. The patients were phenotyped as either MHS or MHN by the caffeine halothane contracture test (CHCT). The CHCT was performed according to a standardized protocol established by the North American MH group. 13
The CHCT results and clinical profile of MHS patients are summarized in table 1. Mean age at the time of enrollment in the study was 27.9 yr (range, 19–49 yr), 23.8 yr (range, 6–53 yr), and 33.8 yr (range, 20–45 yr) for MHS, MHN, and control groups, respectively. Genetic screening of mutations in RYR1 of these patients was previously performed. 10The mutations tested in the study were Cys35Arg , Arg163Cys , Gly248Arg , Gly341Arg , Ile403Met , Arg552Trp , Arg614Cys , Arg614Leu , Arg2163Cys , Arg2163His , Val2168Met , Val2214Ile , Ala2367Thr , Asp2431Asn , Gly2434Arg , Arg2435His , Arg2454Cys , Arg2454His , Arg2458Cys , Arg2458His , and Ile4898Thr . Among 13 MHS patients, 5 patients were found to carry a mutation in RYR1 (table 1). The remaining nine MHS patients were negative for the 21 RYR1 mutations tested.
Ca2+Mobilization Test Using B Cells
Relative changes in [Ca2+]iin B cells were derived from changes in the fluorescence intensity of fluo-3-loaded cells. 14Peripheral mononuclear cells (PMCs) were isolated from 25-ml blood samples by Ficoll-Hypaque density gradient centrifugation. Cells (2 × 106/ml) were loaded with 1 μm acetoxy-methyl ester of fluo-3 (Molecular Probes, Eugene, OR) by incubation in subdued light (30 min; 25°C). No difference in the viability of the cells was observed between MHS and MHN (or control) individuals. The resultant fluo-3-loaded cells were then stained with phycoerythrin (PE)-conjugated anti-CD19 mAB to selectively label B cells. Cells were then washed three times with Hanks balanced salt solution (HBSS), resuspended in 1 ml HBSS and analyzed by FACScan (Becton-Dickinson, San Jose, CA). Forward and right-angle scatter signals were displayed on a linear scale, with the forward scatter adjusted to gate cells from debris. For dual-color analysis of intracellular fluo-3 and surface-labeled PE, the fluo-3-fluorescence (excitation at 488 nm with emission at 525 nm) and PE (excitation at 488 nm, with emission at 585 nm) signals were detected after separation with 530 (FL-1) and 585 nm (FL-2) bandpass filters, respectively. FL-1 fluorescence and FL-2 fluorescence are recorded, amplified, and displayed on a logarithmic scale. Crossover of FL-1 fluorescence into the FL-2 detection window was compensated for by analog subtraction at the preamplifier stage. For each experiment, the fluo-3-loaded cells were analyzed to obtain an unstimulated baseline. Cells were then exposed to caffeine or 4CmC and analyzed every 15–30 s at rates of 400–1,000 cells/s. Transit time required for data acquisition was approximately 10–20 s. The measurement was done at room temperature. To analyze fluo-3 fluorescence shift of PE-labeled cells, FL-1 histograms of FL-2+cells were obtained by gating FL-2–positive clusters in a FL-1 versus FL-2 dot plot display. The percentage of fluo-3+cells relative to unstimulated baseline was then calculated and analyzed using Consort 30 program (Becton-Dickinson). Neither caffeine nor 4CmC affected fluo-3 fluorescence properties in this system. In some experiments, time-dependent continuous acquisition and analysis were performed using CellQuest software (Becton-Dickinson). For dose-response experiments, changes in [Ca2+]iwere examined after 25 and 50 mm caffeine and after 100, 200, and 400 μm 4CmC. The larger populations were then tested for 50 mm caffeine and 400 μm 4CmC. In a study of a family with the Val2168Met mutation, 600 μm 4CmC was tested in addition to 200 and 400 μm 4CmC.
Selective Reverse Transcription-Polymerase Chain Reaction Followed by Restriction Fragment Length Polymorphism Analysis
Expression of the RYR1 messenger RNA (mRNA) in B cells was examined as follows. Because only small number of cells were available for RNA isolation after the Ca2+assay, B cells were not isolated from PMCs since we have shown that RYR1 mRNA from PMCs are mainly from B cells. 15Total RNA was extracted using SV Total RNA Isolation System (Promega, Madison, WI); reverse transcription was performed to the first strand of cDNA using a cDNA synthesis kit (Promega). Synthesized cDNA was then amplified by reverse transcription (RT)-polymerase chain reaction (PCR). The PCR amplifications were carried out using Expand Long PCR system (Boehringer Mannheim, Indianapolis, IN), using a primer set that amplifies the exon 39 portion of the RYR1 , forward primer; 5′-dCGTGGAAGACACCATGAGCCTGCT-3′ and reverse primer; 5′-ACCCGATGCTCTGGATCATGA-3′. PCR was performed in a 50-μl reaction mixture containing 100 ng cDNA, 15 pmol of each primer, 0.5 mm dNTP, 2.5 U Expand Long polymerase mixture and Expand Long PCR buffer 3 (Boehringer Mannheim). The PCR amplification conditions were 95°C for 2 min, followed by 35 cycles of 95°C for 1 min, 58°C for 2 min, and 68°C for 3 min, followed by a 7-min extension at 68°C. The RT-PCR products were then digested using a restriction enzyme, BsgI, to identify G6502A substitution. Based on the known sequences of human RYR isoforms, Bsg I cuts the amplified 113 base pairs (bp) RYR1 product into 86- and 27-bp fragments. The restriction fragments were then resolved by electrophoresis on a 2% agarose gel and visualized on a UV transilluminator. As a control of mRNA input, β-actin mRNA concentrations were determined for each sample in separate RT-PCR reactions. For β-actin amplification, PCR was performed with 25 cycles to ensure that the amplification was completed within the linear range. The sequences of primers for β-actin were 5′-dAAGAGAGGCATCCTCACCCT-3′ (sense) and 5′-dTGCTGATCCACATCTGCTGGA-3′ (antisense). The signal ratio of RYR1 to β-actin was determined on the basis of the ratio of the intensity of the PCR product compared with the corresponding β-actin band. The PCR products were imaged and the relative optical density of each band was measured and analyzed using NIH Image software (National Institutes of Health, Bethesda, MD).
Nested Polymerase Chain Reaction Followed by Restriction Fragment Length Polymorphism
The RYR1 mRNA expressed in B cells from members of a family with the Val2168Met mutation was also tested for the mutation following nested PCR. Nested PCR is a two-step PCR that amplifies an internal target by the second PCR from longer PCR products obtained by the initial PCR. This technique eliminates the possibility of amplification from contaminated undigested genomic DNA in the cDNA samples. Thus, the 1.2-kilobase pair (kbp) sequence consisted of multiple exons (exons 39–47) was first amplified so that the cDNA-derived amplicons (1.2 kbp) could be selectively obtained by separating genomic-derived amplicons (> 6 kbp) on gel electrophoresis. The RYR1 was amplified using primers (upstream primer; 5′-dTGGGCCCAAGAGGACTTCGT-3′, downstream primer; 5′-dAGCACCATGGACGCCTTGTG-3′). The PCR amplification conditions were 95°C for 2 min, followed by 30 cycles of 95°C for 1 min, 58°C for 2 min and 68°C for 3 min, followed by a 7-min extension at 68°C. The RT-PCR products were resolved by electrophoresis on a 1% agarose gel and visualized on a UV transilluminator. A PCR product of approximately 1,200 bp was purified from a 1% agarose gel with QIAquick gel extraction kit (QIAGEN, Valencia, CA). The 1.2-kbp amplicon was then tested for Val2168Met (G6502A) by PCR-based restriction fragment length polymorphism as described previously (Selective Reverse Transcription-Polymerase Chain Reaction Followed by Restriction Fragment Length Polymorphism Analysis).
Kruskal-Wallis and Mann-Whitney tests were used for nonparametric evaluations, where applicable. Dose-response data for 4CmC were analyzed using repeated measures (RM) analysis of variance (ANOVA).
Ca2+Responses of B Lymphocytes to Caffeine and 4CmC
The difference in the Ca2+response in B cells between MHS (n = 6) individuals and controls (n = 6) was statistically significant following addition of 50 mm caffeine (33.0 ± 3.6 vs. 15.5 ± 3.1, P < 0.005) but not following 25 mm caffeine (8.5 ± 1.7 vs. 3.4 ± 2.0, P > 0.05). The representative results from two pairs of MHS and control individuals for Ca2+response to caffeine were shown in figures 1A and B. Differences in 4CmC-induced changes in [Ca2+]ibetween the three groups were greater at 400 μm than at 200 or 100 μm (fig. 1C). The RM ANOVA test at 400 μm indicated a significance of P = 0.010. The difference at 200 μm indicated a P = 0.042, while there was no difference at 100 μm. Based on the results obtained from the dose-response experiments, concentrations of 50 and 400 μm were chosen to test for the effects of caffeine and 4CmC on B cells.
The Ca2+response of B cells to caffeine (50 mm) and 4CmC (400 μm) in 13 MHS individuals (11 MHS individuals for 4CmC), 21 MHN individuals and 18 controls are shown in figure 2. Caffeine-induced Ca2+response significantly differed between the MHS, MHN, and control groups (P = 0.0001, Kruskal-Wallis test). Caffeine-induced (50 mm) increases in [Ca2+]iin B cells were significantly greater in MHS than in MHN (P = 0.0004), control (P = 0.0001), and non-MHS individuals (MHN plus control, P < 0.0001) by Mann-Whitney test. Similarly, 4CmC-induced (400 μm) Ca2+responses significantly differ among the three groups (P = 0.028). Mann-Whitney test indicated that 4CmC-induced (400 μm) increases in [Ca2+]iwere significantly greater in MHS individuals than in controls (P = 0.003) or non-MHS individuals (MHN individuals plus controls, P = 0.0078). The nonparametric Spearman rank correlation test indicated a significant correlation between Ca2+response to caffeine (50 mm) and 4CmC (400 μm) (fig. 2C) (r = 0.608, P < 0.0001).
Expression of the RYR1 Mutant and Ca2+Response in B Cells in the Family with the Val2168Met Mutation
The CHCT and genotype results of a family with the Val2168Met mutation were reported previously. 10Correlation between muscle phenotype and genotype was seen in this family (fig. 3A). The referred patient was found to be positive for MHS by CHCT. That individual's parent and sibling also had positive CHCT results in 1981 and 1988, respectively. A maternal sibling died during anesthesia under circumstances consistent with MH. All three CHCT-positive individuals in this family had Val2168Met (G6502A), while the CHCT-negative relatives had no mutation in RYR1 .
The 4CmC caused a dose-dependent increase in [Ca2+]iin B cells from this family (fig. 3B). ANOVA revealed a significant MH status by 4CmC concentration interaction (P = 0.004), indicating that 4CmC-induced increases in [Ca2+]iover concentration depend on the MHS status. The 4CmC-induced Ca2+increases were significantly greater at 600 μm in the three MHS members than in the other members (%fluo-3+cells; 39.3 ± 5.5 and 17.6 ± 3.3%, P = 0.028). It was also noted that all three MHS individuals showed increases in [Ca2+]iat 200 μm from unstimulated basal concentrations, while the mutation-negative members exhibited no increase, indicating a lower threshold to 4CmC in the MHS B cells in the family. Caffeine-induced (50 mm) increases in [Ca2+]iin mutation-positive members were not different from those in mutation-negative members (%fluo-3+cells; 16.4 ± 3.2 and 19.8 ± 5.0%, P > 0.05).
Expression of the RYR1 mRNA in B cells was examined using semiquantitative RT-PCR (fig. 3C). The B cells from all individuals expressed RYR1 mRNA (fig. 3C). Relative levels of the RYR1 expression were determined by comparing with levels of a housekeeping gene β-actin (fig. 3C) and expressed as the signal ratio of RYR1 to β-actin (fig. 3C). There was a tendency for the high 4CmC responders (MHS) to have higher expression levels of RYR1 , but overall results showed no clear relation between the 4CmC Ca2+response and RYR1 expression in the family members (RYR1 /β-actin ratio, MHS vs. MHN; 0.85 ± 0.15 vs. 0.52 ± 0.20%, P > 0.05;fig. 3C).
Finally, we examined whether the Val2168Met (G6502A) mutant was expressed in B cells. DNase-treated RNA isolated from B cells was tested for the mutation. Consistent with the results obtained from genomic DNA, the mutation-positive members (II-1, III-1, and III-3) expressed mutant RNA in B cells, while I-2, II-1, and III-2 expressed normal RNA (fig. 3D).
Previously, we reported that RYR1 was expressed in human B lymphocytes and functioned as a Ca2+-release channel during B-cell receptor-stimulated Ca2+signaling process. 12In the present study, we tested 52 unrelated individuals (13 MHS individuals, 21 MHN individuals, and 18 controls) and 6 members of a single family for the Ca2+response of B cells to RYR-stimulating agents caffeine or 4CmC using flow cytometric dual-color analysis. The study showed that Ca2+responses induced by caffeine and 4CmC in B cells from MHS patients are larger than the responses from MHN and control individuals. We also show that B cells express mutant mRNA of RYR1 in patients with the Val2168Met mutation (G6502A) and that the B-cell Ca2+phenotype segregates with genotype and CHCT results in a family with this mutation. These results suggest that the B-cell Ca2+phenotype may be altered in MHS individuals and that the alterations are associated with mutations in the RYR1 gene in some MHS individuals. This type of study may help in studying the genotype-phenotype association in patients with various MH mutations.
Pharmacologic characterization of effects of caffeine and 4CmC on Ca2+response in B cells suggests that a key protein in the heightened Ca2+response in MHS individuals is RYR1 or possibly proteins associated with the RYR1 . 4CmC has been shown to induce Ca2+release from a ruthenium red/caffeine-sensitive Ca2+release channel in skeletal muscle, from heparin-treated cerebellar microsomes and from IP3-insensitive pools in PC12 cells. 16These findings suggest that 4CmC selectively activates RYR-mediated Ca2+release in different types of cells. Similarly, in B cells, 4CmC activates RYR1 -mediated Ca2+release, as shown by its ability to release Ca2+after depletion of IP3-sensitive pools by cross-linking membrane immunoglobulin M. 12The dose-responses for 4CmC suggested a lower threshold for 4CmC-induced Ca2+release in MHS individuals than in MHN or control individuals. The result was consistent with the 4CmC dose-response results from the skeletal muscle. 17
In contrast to 4CmC, which appears to activate mainly Ca2+release in B cells, we found that caffeine induced Ca2+influx from the extracellular compartment. 18Thus, the caffeine-induced increase in [Ca2+]iwas abolished in EGTA-treated or Ca2+-free media. 18At concentrations of 1–50 mm, caffeine causes an increase in [Ca2+]iin a concentration-dependent fashion. 18The increase in [Ca2+]ibecomes apparent at the high concentration of 25 mm. 18Requirement of more than 20 mm caffeine for activating Ca2+response is not unusual. In nonmuscle cells, such as neurons and chromaffin cells, concentrations as high as 40–50 mm are often required to obtain maximal Ca2+response. 19,20Although the nature of the Ca2+channel and the mechanism whereby caffeine elicits opening are currently unknown, our previous studies suggest that caffeine activates nonselective cation channels in B cells. 18Thus, the Ca2+channel activated by caffeine is Mn2+permeable, and is insensitive to a store-operated Ca2+channel (SOC) blocker SKF-96365. 18We hypothesize that caffeine induces Ca2+influx via a nonselective cation channel and then Ca2+activates a RYR1 -mediated opening of SOC, as has been demonstrated in RYR1 -transfected HEK 293 cells. 21In this hypothesis, the caffeine-induced Ca2+response involves RYR1 and can be substantially enhanced in MHS individuals who have mutations in the RYR1 . Alternatively, there is a common mechanism in caffeine and 4CmC-induced Ca2+response that is likely mediated by RYR1 , as suggested by a positive correlation between the two measures (fig. 2C). However, the caffeine-induced Ca2+response likely involves other cellular factors and thus could be more sensitive but less specific to RYR1 -related mechanism than is the 4CmC-induced Ca2+response. This may account for greater differences between the groups in the caffeine-induced Ca2+response than in 4CmC-induced responses. It should be stressed that other components that modulate the caffeine-induced Ca2+phenomenon may be relevant to other causal genes for MH.
There was a segregation of Val2168Met (G6502A) with a CHCT phenotype and B-cell Ca2+response in a family of three generations. In this family, the B cells from MHS patients express the RYR1 mutants and abnormal Ca2+response and thus verified our rationale of testing B cells for the RYR-stimulating agents. Moreover, expression of RYR1 was confirmed for all the members tested for the Ca2+response. Therefore, the Ca2+results were valid for evaluating the status of RYR1 for every family member. Alternatively, if RYR1 was not expressed, low caffeine- or 4CmC-induced Ca2+responses could not be explained by altered function of the RYR1 . Consistent with dose-response data (fig. 1C), the dose-response results from this family indicated that all mutation-positive members showed increases in Ca2+at 200 μm from the basal concentrations, whereas mutation-negative members did not. This is consistent with observations that the MHS skeletal muscle showed a lower threshold to 4CmC than did MHN muscle. 17In this family study, we have tested 600 μm 4CmC for Ca2+response. The dose-response experiments indicated a clearer separation of MHS individuals from MHN individuals at 600 μm 4CmC than was obtained at 400 μm. Although the concentration 400 μm was initially applied to compare the larger populations for the Ca2+response to 4CmC (fig. 2B), the results from this family study suggest that 600 μm 4CmC would give better separation of MHS from MHN than 400 μm. There was no difference in caffeine-induced Ca2+response between mutation-positive and -negative members in this family. The main reason was that caffeine failed to enhance Ca2+response in mutation-positive members. Thus, Ca2+response to caffeine should be further examined to study a mechanism of lack in enhancement in these mutation-positive individuals.
Phenotyping of patients as either MHS or MHN has been performed by a procedure involving muscle biopsy of the vastus lateralis, followed by in vitro CHCT, in North America, and in vitro contracture test, in Europe. 13The mechanism of these contracture tests is thought to depend on the abnormal Ca2+regulation mediated by RYR-1 or associated molecules in muscle from MHS compared with MHN individuals. Altered Ca2+homeostasis in B cells in MHS individuals, similar to the alteration demonstrated for muscle cells, increases the possibility of a minimally invasive blood test. A scattergram showing Δ% responding cells (fig. 2A and B) reveals significant differences, but with imperfect discrimination between MHS, MHN, and control groups for the Ca2+response. It is apparent that heterogeneity of MH genotype would at least complicate discrimination between MHS and MHN by B-cell Ca2+phenotype. Further studies will determine whether Ca2+signaling in B cells can be used for reliable prediction of MHS.
The enhanced Ca2+response to these RYR-stimulating agents, which mimics that of skeletal muscle from MHS patients, suggests that B cells and skeletal muscles share a common mechanism of controlling [Ca2+]i, which is conditionally hyperresponsive to RYR-stimulating agents. Thus, although the validity of the test was not proven in this study, B-cell Ca2+phenotyping may be useful in the future as a complementary test to CHCT. It should be also stressed that RYR1 mRNA expressed in B cells can be used for effective screening of mutations in the RYR1 gene and also for gene transfection studies. Moreover, studying the mechanism of Ca2+homeostasis in B cells may provide a unique opportunity to investigate molecular mechanisms of MH and possibly to find other candidate genes causal for MH.
The authors thank Drs. Anthony S. Basile, Ph.D. (Senior Investigator, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland), Rulph Bunger, Ph.D. (Professor, Uniformed Services University of the Health Sciences, Maryland), Tommy McCarthy Ph.D. (Professor, University College, Cork, Ireland), Leslie McKinney, Ph.D. (Assistant Professor, Uniformed Services University of the Health Sciences, Maryland), Paul Mongan, M.D. (Associate Professor, Uniformed Services University of the Health Sciences, Maryland), Thomas E. Nelson, Ph.D. (Professor, Wake Forest University School of Medicine), Henry Rosenberg, M.D. (Professor, Thomas Jefferson University-Jefferson Medical College, Philadelphia, Pennsylvania), Shizuko Sei, M.D. (Laboratory Chief, NCI at Frederick, Maryland) and Frank Wappler, M.D. (Professor, University Hospital Eppendorf, Hamburg, Germany) for helpful discussions and critical comments.