An electron paramagnetic resonance study of skeletal muscle membrane fluidity in malignant hyperthermia

Skeletal muscle sarcolemma (SL), transverse tubule (TT) and heavy sarcoplasmic reticulum (HSR) membranes were isolated from malignant hyperthermia susceptible (MHS) and normal pigs, and the rotational dynamics of lipid hydrocarbon chain motion was examined by electron paramagnetic resonance (EPR) spectroscopy. The stearic acid spin probe 16-SASL was incorporated into MHS and normal membranes and both the order parameter (S) and effective correlation time (tau r) of probe motion were calculated from spectra recorded over the temperature range of 2 to 40 degrees C. At any given temperature, TT membranes exhibited significantly greater values for both the S and tau r of probe motion than did SL, which exhibited significantly greater values than did HSR membranes. The order of decreasing S and tau r values for 16-SASL mobility correlated with the decreasing cholesterol content of these membranes (TT greater than SL greater than HSR), however there was no difference in the S or tau r values for a given membrane fraction isolated from both MHS and normal muscle. Arrhenius plots of 16-SASL mobility in SL, TT and HSR were linear from 2 to 40 degrees C, indicating no abrupt thermotropic change in the lipid hydrocarbon phase of any of the membrane types studied. Apparent activation energies (Ea), calculated from the Arrhenius plots, were similar for MHS and normal membranes derived from a given cellular location. However, the Ea of probe motion for TT membranes (2.3 +/- 0.1 and 2.4 +/- 0.1 kcal/mol/degree for MHS and normal, respectively) was significantly less than for SL (3.4 +/- 0.4 and 2.9 +/- 0.1 kcal/mol/degree for MHS and normal, respectively) which, in turn, was significantly less than the Ea for HSR (3.7 +/- 0.1 and 3.7 +/- 0.1 kcal/mol/degree for MHS and normal, respectively). Since 16-SASL motion was similar in MHS and normal membranes, we conclude that there is no evidence for a generalized membrane defect affecting lipid mobility in these MHS muscle membranes.     

Voltage-dependent calcium release in human malignant hyperthermia muscle fibers.

Malignant hyperthermia (MH) results from a defect of calcium release control in skeletal muscle that is often caused by point mutations in the ryanodine receptor gene (RYR1). In malignant hyperthermia-susceptible (MHS) muscle, calcium release responds more sensitively to drugs such as halothane and caffeine. In addition, experiments on the porcine homolog of malignant hyperthermia (mutation Arg615Cys in RYR1) indicated a higher sensitivity to membrane depolarization. Here, we investigated depolarization-dependent calcium release under voltage clamp conditions in human MHS muscle. Segments of muscle fibers dissected from biopsies of the vastus lateralis muscle of MHN (malignant hyperthermia negative) and MHS subjects were voltage-clamped in a double vaseline gap system. Free calcium was determined with the fluorescent indicator fura-2 and converted to an estimate of the rate of SR calcium release. Both MHN and MHS fibers showed an initial peak of the release rate, a subsequent decline, and rapid turn-off after repolarization. Neither the kinetics nor the voltage dependence of calcium release showed significant deviations from controls, but the average maximal peak rate of release was about threefold larger in MHS fibers.     

Inhibition of sarcoplasmic Ca2+-ATPase increases caffeine- and halothane-induced contractures in muscle bundles of malignant hyperthermia susceptible and healthy individuals

Background

Malignant hyperthermia (MH) is triggered by halogenated anaesthetics and depolarising muscle relaxants, leading to an uncontrolled hypermetabolic state of skeletal muscle. An uncontrolled sarcoplasmic Ca²⁺ release is mediated via the ryanodine receptor. A compensatory mechanism of increased sarcoplasmic Ca²⁺-ATPase activity was described in pigs and in transfected cell lines. We hypothesized that inhibition of Ca²⁺ reuptake via the sarcoplasmic Ca²⁺-ATPase (SERCA) enhances halothane- and caffeine-induced muscle contractures in MH susceptible more than in non-susceptible skeletal muscle.

Methods

With informed consent, surplus muscle bundles of 7 MHS (susceptible), 7 MHE (equivocal) and 16 MHN (non-susceptible) classified patients were mounted to an isometric force transducer, electrically stimulated, preloaded and equilibrated. Following 15 min incubation with cyclopiazonic acid (CPA) 25 μM, the European MH standard in-vitro-contracture test protocol with caffeine (0.5; 1; 1.5; 2; 3; 4 mM) and halothane (0.11; 0.22; 0.44; 0.66 mM) was performed. Data as median and quartiles; Friedman- and Wilcoxon-test for differences with and without CPA; p < 0.05.

Results

Initial length, weight, maximum twitch height, predrug resting tension and predrug twitch height of muscle bundles did not differ between groups. CPA increased halothane- and caffeine-induced contractures significantly. This increase was more pronounced in MHS and MHE than in MHN muscle bundles.

Conclusion

Inhibition of the SERCA activity by CPA enhances halothane- and caffeine-induced contractures especially in MHS and MHE skeletal muscle and may help for the diagnostic assignment of MH susceptibility. The status of SERCA activity may play a significant but so far unknown role in the genesis of malignant hyperthermia.     

Evidence for genetic heterogeneity of malignant hyperthermia susceptibility

A locus for malignant hyperthermia susceptibility (MHS) has been localized on chromosome 19q12-13.2, while at the same time the gene encoding the skeletal muscle ryanodine receptor (RYR1) also has been mapped to this region and has been found to be tightly linked to MHS. RYR1 was consequently postulated as the candidate for the molecular defect causing MHS, and a point mutation in the gene has now been identified and is thought to be the cause of MH in at least some MHS patients. Here we report the results of a linkage study done with 19q12-13.2 markers, including the RYR1 cDNA, in two Bavarian families with MHS. In one of the families, three unambiguous recombination events between MHS and the RYR1 locus were found. In the second family only one informative meiosis was seen with RYR1. However, segregation analysis with markers for D19S75, D19S28, D19S47, CYP2A, BCL3, and APOC2 shows that the crossovers in the first family involve the entire haplotype defined by these markers flanking RYR1 and, furthermore, reveals multiple crossovers between these haplotypes and MHS in the second family. In these families, pairwise and multipoint lod scores below -2 exclude MHS from an interval spanning more than 26 cM and comprising the RYR1 and the previously described MHS locus. Our findings thus strongly suggest genetic heterogeneity of the MHS trait and prompt the search for another MHS locus.     

Enhanced response to caffeine and 4-chloro-m-cresol in malignant hyperthermia-susceptible muscle is related in part to chronically elevated resting [Ca2+]i

Malignant hyperthermia (MH) is a potentially fatal pharmacogenetic syndrome caused by exposure to halogenated volatile anesthetics and/or depolarizing muscle relaxants. We have measured intracellular Ca²⁺ concentration ([Ca²⁺]_i) using double-barreled, Ca²⁺-selective microelectrodes in myoballs prepared from skeletal muscle of MH-susceptible (MHS) and MH-nonsusceptible (MHN) swine. Resting [Ca²⁺]_i was approximately twofold in MHS compared with MHN quiescent myoballs (232 ± 35 vs. 112 ± 11 nM). Treatment of myoballs with caffeine or 4-chloro-m-cresol (4-CmC) produced an elevation in [Ca²⁺]_i in both groups; however, the concentration required to cause a rise in [Ca²⁺]_i elevation was four times lower in MHS than in MHN skeletal muscle cells. Incubation of MHS cells with the fast-complexing Ca²⁺ buffer BAPTA reduced [Ca²⁺]_i, raised the concentration of caffeine and 4-CmC required to cause an elevation of [Ca²⁺]_i, and reduced the amount of Ca²⁺ release associated with exposure to any given concentration of caffeine or 4-CmC to MHN levels. These results suggest that the differences in the response of MHS skeletal myoballs to caffeine and 4-CmC may be mediated at least in part by the chronic high resting [Ca²⁺]_i levels in these cells. calcium homeostasis; 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid     

Abnormal fluidity state in membranes of malignant hyperthermia pig skeletal muscle

The fluidity state was analyzed on sarcoplasmic reticulum membranes and phospholipid vesicles prepared from normal and malignant hyperthermia susceptible pig muscle. Electron spin resonance studies were performed to determine the fluidity state at the region near the polar headgroups and in the central core of the bilayer using 5-nitroxide (5-NS) and 16-nitroxide stearic acid (16-NS), respectively. With the 5-NS label, no differences were found between normal and malignant hyperthermia sarcoplasmic reticulum (MH SR) membranes whereas with the 16-NS label, a significant increase of the activation energy was shown with MH membranes. Lower values of fluorescence anisotropy observed with DPH-labeled MH membranes as compared with normal ones, confirmed the higher abnormal fluidity state of these membranes. The fluidizing effect of halothane, a triggering agent of malignant hyperthermia syndrome, was also studied in these membranes. We show that a relatively low concentration of the drug destabilized not only the diseased sarcoplasmic reticulum membranes but also the vesicles made of total phospholipids extracted from MH skeletal muscle. Together, these findings strongly suggest that an overall increase in membrane fluidity may be implied in the MH disease, improving the general membrane defect hypothesis for this syndrome.     

Erythrocyte membrane ATPase and calcium pumping activities in porcine malignant hyperthermia

To investigate possible abnormalities in erythrocyte membrane enzyme activities in the pharmacogenetic disorder MH, membrane ATPase activities have been examined in erythrocyte ghosts prepared from red blood cells of MHS and normal swine. While no differences were noted in Mg2+-ATPase activities, the (Na+, K+)-ATPase activity of MHS erythrocyte ghosts was less than that of normal ghosts. Ca2+-ATPase activity exhibited low- and high-affinity Ca2+-binding sites in both types of erythrocyte ghost. While the Km for Ca2+ was greater for normal than for MHS erythrocyte ghosts at the high-affinity Ca2+-binding site, the reverse was true at the low-affinity Ca2+-binding site. Irrespective of the type of calcium binding site occupied, the Vmax for normal erythrocyte ghost Ca2+-ATPase activity was greater than that for MHS ghosts. In the presence of calmodulin, there was now no difference between MHS and normal erythrocyte ghosts in either the Km for Ca2+ or the Vmax of the Ca2+-ATPase activity. To determine if the calcium pumping activity of intact MHS and normal pig erythrocytes differed, calcium efflux from the 45Ca-loaded erythrocytes was determined; this activity was significantly greater for MHS than for normal erythrocytes. Thus, the present study confirms that there are abnormalities in the membranes of MHS pig red blood cells. However, we conclude that these abnormalities are unlikely to result in an impaired ability of MHS erythrocytes to regulate their cytosolic Ca2+ concentration.     

Distinct immunopeptide maps of the sarcoplasmic reticulum Ca2+ release channel in malignant hyperthermia

Sarcoplasmic reticulum isolated from malignant hyperthermia-susceptible (MHS) muscle exhibits abnormalities in the regulation of calcium release. To identify the molecular basis of this abnormality, the Ca2+ release channel from both normal and MHS sarcoplasmic reticulum was examined using proteolytic digestion followed by immunoblot staining with a polyclonal antibody against the rabbit Ca2+ release channel protein. Under appropriate conditions, trypsin digestion of isolated sarcoplasmic reticulum vesicles from the two types of pigs revealed a distinct difference in the immunostaining pattern of the Ca2+ release channel-derived peptides. An approximate 86-kDa peptide was the predominant fragment in normal sarcoplasmic reticulum while an approximate 99-kDa peptide fragment was the major peptide detected in MHS sarcoplasmic reticulum. Digestion of sarcoplasmic reticulum vesicles isolated from four normal and four MHS pigs showed that the differences were highly reproducible. Trypsin digestion of sarcoplasmic reticulum isolated from heterozygous pigs, which contain one normal and one MHS allele, showed an antibody staining pattern that was intermediate between MHS and normal sarcoplasmic reticulum. These results can be explained by a primary amino acid sequence difference between the normal and MHS Ca2+ release channels and support the hypothesis that a mutation in the gene coding for the sarcoplasmic reticulum Ca2+ release channel is responsible for malignant hyperthermia.     

Ca2+ Influx via the Na+/Ca2+ Exchanger Is Enhanced in Malignant Hyperthermia Skeletal Muscle

Malignant hyperthermia (MH) is potentially fatal pharmacogenetic disorder of skeletal muscle caused by intracellular Ca²⁺ dysregulation. NCX is a bidirectional transporter that effluxes (forward mode) or influxes (reverse mode) Ca²⁺ depending on cellular activity. Resting intracellular calcium ([Ca²⁺]_r) and sodium ([Na⁺]_r) concentrations are elevated in MH susceptible (MHS) swine and murine muscles compared with their normal (MHN) counterparts, although the contribution of NCX is unclear. Lowering [Na⁺]_e elevates [Ca²⁺]_r in both MHN and MHS swine muscle fibers and it is prevented by removal of extracellular Ca²⁺ or reduced by t-tubule disruption, in both genotypes. KB-R7943, a nonselective NCX3 blocker, reduced [Ca²⁺]_r in both swine and murine MHN and MHS muscle fibers at rest and decreased the magnitude of the elevation of [Ca²⁺]_r observed in MHS fibers after exposure to halothane. YM-244769, a high affinity reverse mode NCX3 blocker, reduces [Ca²⁺]_r in MHS muscle fibers and decreases the amplitude of [Ca²⁺]_r rise triggered by halothane, but had no effect on [Ca²⁺]_r in MHN muscle. In addition, YM-244769 reduced the peak and area under the curve of the Ca²⁺ transient elicited by high [K⁺]_e and increased its rate of decay in MHS muscle fibers. siRNA knockdown of NCX3 in MHS myotubes reduced [Ca²⁺]_r and the Ca²⁺ transient area induced by high [K⁺]_e. These results demonstrate a functional NCX3 in skeletal muscle whose activity is enhanced in MHS. Moreover reverse mode NCX3 contributes to the Ca²⁺ transients associated with K⁺-induced depolarization and the halothane-triggered MH episode in MHS muscle fibers.     

Evidence for linkage of the central core disease locus to the proximal long arm of human chromosome 19

Central core disease of muscle (CCD; MIM 117000) is a rare inheritable myopathy that is frequently found in association with susceptibility to malignant hyperthermia (MHS). This observation has prompted us to perform a linkage study in CCD families using various chromosome 19q probes that are linked to the MHS locus and map close to the ryanodine receptor gene (RYR1), a strong MHS candidate gene. Our genetic linkage data support a location of the CCD gene on proximal 19q13.1 and thus suggest that CCD and MHS may be allelic.     

High-resolution physical mapping of four microsatellite repeat markers near the RYR1 locus on chromosome 19q13.1 and apparent exclusion of the MHS locus from this region in two malignant hyperthermia susceptible families.

Malignant hyperthermia susceptibility (MHS) is a potentially lethal, hereditary disorder of skeletal muscle that may be triggered by inhalation anesthetics and depolarizing muscle relaxants. Defects in the gene encoding the ryanodine receptor (RYR1) localized on human chromosome 19q13.1 have been proposed to be responsible for MHS. Using a chromosome 19-specific human/hamster somatic cell hybrid mapping panel, we were able to determine that four closely linked microsatellite repeat markers bracket RYR1 with the order 19cen-D19S75-D19S191-RYR1-(D19S47, D19S190)-19ter. Application of the four markers to genetic studies of MHS showed recombination between the markers and MHS in two families, with linkage analysis apparently excluding the MHS locus from the RYR1 region of 19q13.1. These results therefore support the recent observations of genetic heterogeneity in MHS.     

Stimulation and inhibition of [3H]ryanodine binding to sarcoplasmic reticulum from malignant hyperthermia susceptible pigs

When compared to normal pig sarcoplasmic reticulum (SR), SR from malignant hyperthermia susceptible (MHS) porcine skeletal muscle has been shown to exhibit an increased rate of calcium release, as well as alterations in [3H]ryanodine-binding activity in the presence of microM Ca2+ (Mickelson et al., 1988, J. Biol. Chem. 263, 9310). In the present study, various stimulators (adenine nucleotides and caffeine) and inhibitors (ruthenium red and Mg2+) of the SR calcium release channel were examined for effects on MHS and normal SR [3H]ryanodine binding. The apparent affinity of the MHS SR receptor for ryanodine in the presence of 10 mM ATP (Kd = 6.0 nM) or 10 mM caffeine (Kd = 28 nM) was significantly greater than that of the normal SR (Kd = 8.5 and 65 nM in 10 mM ATP or caffeine, respectively), the Bmax (12-16 pmol/mg) was similar in all cases. The Ca2+(0.5) for inhibition of [3H]ryanodine binding in the presence of 5 mM AMPPNP (238 vs 74 microM for MHS and normal SR, respectively) and the Ca2+(0.5) for stimulation of [3H]ryanodine binding in the presence of 5 mM caffeine (0.049 vs 0.070 microM for MHS and normal SR, respectively) were also significantly different. Furthermore, in the presence of optimal Ca2+, MHS SR [3H]ryanodine binding was more sensitive to caffeine stimulation (C0.5 of 1.7 vs 3.4 mM) and was less sensitive to ruthenium red (C0.5 of 1.9 vs 1.2 microM) or Mg2+ inhibition (C0.5 of 0.34 vs 0.21 mM) than was normal SR. These results further support the hypothesis that differences in the ryanodine/receptor calcium release channel regulatory properties are responsible for the abnormal calcium releasing activity of MHS SR.     

Transverse tubule calcium regulation in malignant hyperthermia

Transverse tubule (TT) calcium transport and permeability were examined in the inherited skeletal muscle disorder malignant hyperthermia (MH). ATP-dependent calcium uptake by TT vesicles isolated from normal and MH-susceptible (MHS) pig muscle had a similar dependence on ionized Ca2+ concentration (K1/2 for Ca2+ of 0.21 +/- 0.04 and 0.25 +/- 0.05 microM for MHS and normal TT, respectively), as well as a similar Vmax (20.9 +/- 2.0 and 23.7 +/- 4.5 nmol Ca/mg protein/min for MHS and normal TT, respectively). Furthermore, the stimulation of calcium uptake by either calmodulin or cAMP-dependent protein kinase was similar in normal and MHS TT. Halothane concentrations greater than 2 mM inhibited calcium uptake by either normal or MHS TT to a similar extent (IC50 = 8 mM). Dantrolene (10 microM), nitrendipine (1 microM), and Bay K 8644 (1 microM) had no significant effect on either the initial rates of calcium uptake or maximal calcium accumulation of either MHS or normal TT vesicles. However, in the absence of any added agents, maximum calcium accumulation by MHS TT was significantly less than by normal TT (90 +/- 10 versus 130 +/- 9 nmol Ca/mg protein after 15 min of uptake). This difference was not due to an increased permeability of MHS TT to calcium, nor was it due to a difference in the sarcoplasmic reticulum contamination (less than 5%) of the MHS and normal preparations. Although our results indicate there is no significant defect in MHS TT calcium regulation, the diminished maximum calcium accumulation by MHS TT may contribute to the abnormal sarcoplasmic calcium homeostasis in skeletal muscle during an MH crisis.     

In vitro effects of sodium fluoride and sodium dichromate on dynamic properties of human erythrocyte membrane

Sodium fluoride (NaF) and sodium dichromate (Na2Cr2O7) are two different toxic compounds which are used as a dental caries prophylactic and as an oxidising agent in various industrial areas, respectively. However, accidental fluoride and chromate poisoning is not a rare occurrence, even death may result from cardiac or respiratory failure. In the present work, alterations produced by NaF, Na2Cr2O7 and temperature changes in the molecular dynamics of the human erythrocyte membrane were studied, in vitro, by the spin-labelling ESR technique. Human intact erythrocyte cells spin labelled with 5- and 16-doxyl stearic acids (5-DSA and 16-DSA) and treated with 40 microM NaF and 5 microM Na2Cr2O7 at 37 degrees C were used to quantify membrane fluidity. This was performed by measuring the changes in the order parameter (S), correlation time (tau) and phase transition temperature using recorded electron spin resonance (ESR) spectra. Experimental results show that 5 microM Na2Cr2O7 and 40 microM NaF do not produce any significant effects on the order parameter of 5-DSA spin label while they cause appreciable changes in the correlation time of the same label. As for 16-DSA, while Na2Cr2O7 does not produce any measurable effect on the order parameter of this label, NaF does in a certain extent. Although weak, the effects of both compounds on the correlation time of 16-DSA are found to be well above the experimental error limits. Change in temperature was observed to alter significantly S and tau parameters which show biphasic character in the temperature range of 5-50 degrees C. Activation energies of the hydrocarbon chains above and below transition temperatures were also determined for untreated and NaF or Na2Cr2O7 treated erythrocyte cells and the effect of NaF and Na2Cr2O7 on these energies and transition temperatures were discussed.     

Allometric dependence of the life span of mammal erythrocytes on thermal stability and sphingomyelin content of plasma membranes

Thermal stability of erythrocyte membrane is a measure for its ability to maintain permeability barrier at deleterious conditions. Hence, it could impact the resistance of erythrocytes against detrimental factors in circulation. In this study the thermostability of erythrocyte membranes was expressed by the temperature, T(go), at which the transmembrane gradient of ion concentration rapidly dissipated during transient heating. T(go) is the inducing temperature of the membrane transition that activated passive ion permeability at hyperthermia causing thermal hemolysis. A good allometric correlation of T(go) to the resistance against thermal hemolysis and the life span of erythrocytes were found for 13 mammals; sheep, cow, goat, dog, horse, man, rabbit, pig, cat, hamster, guinea pig, rat, and mouse. For the same group, the values of T(go) were strictly related to the sphingomyelin content of erythrocyte membranes. The residual ion permeability, P, was temperature activated from 38 to 57 degrees C with activation energy of 250+/-15 kJ/mol that strongly differed from that below 37 degrees C. The projected value of P at 37 degrees C was about half that of residual physiological permeability for Na+ and K+ that build ground for possible explanation of the life span vs membrane thermostability allometric correlation.     

In Vivo 31P NMR Spectroscopy Studies of Halothane Induced Porcine Stress Syndrome. No Effect of C-Phenyl N-Tertbutyl Nitrone (PBN)

Porcine stress syndrome (PSS) which is an example of malignant hyperthermia (MH) in swine has previously been attributed to oxidative stress primarily due to an inherited antioxidant abnormality in MH susceptible (MHS) animals. C-phenyl-N-tert-butyl nitrone (PBN), a free radical spin trap, was selected to investigate whether free radicals are involved in MH. If free radicals cause the MH stress attack, then PBN should alter the time required for the onset of the stress attack, or perhaps protect the animal from experiencing the stress attack. In vivo phosphorus-31 (³¹P) magnetic resonance spectroscopy (MRS) was used to monitor metabolism in three to four week old normal and MHS piglets administered halothane as the stress challenge. Malignant hyperthermia was not reproducibly induced by halothane anesthesia. For those animals which did develop MH a dramatic fall in the level of PCr and a rise in the level of Pi was detected by ³¹P MRS. Intravenous administration of PBN prior to halothane exposure had no effect on the number of animals experiencing the stress attack. PBN does not appear to prevent, delay or reverse the onset of halothane-induced MH in three to four week old MHS piglets. The primary events leading to the MH syndrome do not appear to be influenced by the intervention of the type of free radicals normally trapped by PBN.     

The G1021A substitution in the RYR1 gene does not cosegregate with malignant hyperthermia susceptibility in a British pedigree.

A single base change in the RYR1 gene encoding the skeletal muscle ryanodine receptor (calcium-sensitive calcium-release channel of the sarcoplasmic reticulum), resulting in the substitution of G1021 by A, has been proposed to underlie malignant-hyperthermia (MH) susceptibility in as many as 10% of cases in the European population. As part of our mutation-screening program in MH-susceptible (MHS) individuals, we have investigated this substitution in individuals from 151 unrelated British MHS families and have detected G1021A heterozygotes in 7 families. This mutation was not found in 156 unrelated MH-negative (MHN) individuals. We also examined eight families with central core disease (CCD): the mutation did not occur in any family members of any disease status (affected or unaffected for CCD, MHS, or MHN). In one large family, the G1021A mutation was found but did not show complete cosegregation with MH susceptibility: it occurred in only 7/12 MHS individuals in the kinship, and susceptibility was inherited from parents who were G1021 homozygotes, as well as from parents who were heterozygotes. On the basis of these findings, it is clearly unreliable at present to offer presymptomatic DNA testing for MH status, even in families in which a mutation has been detected.     

Low-pH association of proteins with the membranes of intact red blood cells. I. Exogenous glycophorin and the CD4 molecule

Glycophorin and CD4 proteins are tightly associated with intact human erythrocyte membranes after a short-time incubation at low pH (1-2 min, pH lower than 5, 37 degrees C). Flow cytometry and epifluorescence microscope observations showed that after incubation of red cells with fluorescein isothiocyanate (FITC) labeled glycophorin at pH values lower than 5, the erythrocyte membrane and subsequently formed ghost membranes were fluorescent. Unlabeled glycophorin was reacted with mouse erythrocytes using the same low-pH conditions. Flow cytometry and fluorescence microscopy showed that anti-glycophorin monoclonal antibodies were able to recognize the epitopes of glycophorin associated with the mouse erythrocytes. Kinetic experiments showed that the interaction of FITC-glycophorin with red cell membranes can be monitored by a decrease in the fluorescence intensity. Erythrocyte associated glycophorin was not removed from the membranes after 24 h incubation in human plasma (in vitro, 39 degrees C). A glycoprotein extract containing CD4 was isolated from a T4-lymphoma cell line (CEM). This protein extract was incubated with erythrocytes using the same low-pH conditions. Fluorescently labeled monoclonal antibodies against CD4 stained the red cells after association of CD4 with the membranes. Electron microscopy showed 10 nm immunoglobulin G-coated gold beads associated with CD4-bearing erythrocyte membranes after incubation with anti-CD4 antibodies and then with the gold beads. The potential use of the CD4-erythrocyte complex as a therapeutical agent against acquired immune deficiency syndrome (AIDS) is suggested.     

Viscosity and order in erythrocyte membranes studied with nanosecond fluorometry

The viscosity and the order in the interior of human erythrocyte membranes were investigated by the fluorescence depolarization technique in the nanosecond region with 1,6-diphenyl-1,3,5-hexatriene (DPH). After pulsed excitation with a polarized light, the fluorescence anisotropy ratio of DPH in membranes rapidly decreased and gave a final value (r infinity). The rate of initial decrease and the value of r infinity related to the viscosity in the interior of the membranes and a wobbling angle of DPH which reflects a size of range for the phospholipid motion relating to the order of membrane structure. For normal human erythrocyte membranes the viscosity and the wobbling angle were obtained to be 0.82 poise and 42 degrees, at 37 degrees C. Similar values were obtained for spectrin-free membranes. Hardened membranes by the cross-linking of the cytoskeletal proteins with glutaraldehyde showed a small wobbling angle of 37 degrees, but the viscosity of them was unchanged.     

Dantrolene does not block calcium pulse-induced calcium release from a putative calcium channel in sarcoplasmic reticulum from malignant hyperthermia and normal pig muscle

Calcium pulse additions to isolated SR membranes can cause a reversible efflux of calcium. The threshold level of calcium loading at which calcium efflux occurs is lower for SR membranes isolated from malignant hyperthermia susceptible (MHS) swine. Dantrolene, a unique muscle relaxant, had no effect on threshold calcium load, amounts and rates of calcium release from SR isolated from control and MHS skeletal muscle. It is concluded that the putative calcium channel through which this calcium pulse-induced calcium release mechanism occurs is not affected by dantrolene under these experimental conditions.