Mg2+ dependence of Ca2+ release from the sarcoplasmic reticulum induced by sevoflurane or halothane in skeletal muscle from humans susceptible to malignant hyperthermia.

BACKGROUND: In normal resting muscle, cytosolic Mg(2+) exerts a potent inhibitory influence on the sarcoplasmic reticulum (SR) Ca(2+) release channel (ryanodine receptor, RyR1). Impaired Mg(2+)-regulation of RyR1 has been proposed as a causal factor in malignant hyperthermia (MH). The aim of this study was to compare the effects of cytosolic Mg(2+) on SR Ca(2+) release induced by halothane or sevoflurane in normal (MHN) and MH susceptible (MHS) human skeletal muscle fibres. METHODS: Samples of vastus medialis muscle were obtained from patients under investigation for MH susceptibility. Single fibres were mechanically skinned and perfused with solutions mimicking the intracellular milieu. Changes in [Ca(2+)](i) were detected using fura-2 fluorescence after application of equimolar halothane or sevoflurane. RESULTS: In MHN fibres, concentrations of sevoflurane or halothane as high as 10 mM typically failed to induce SR Ca(2+) release at physiological free [Mg(2+)] (1 mM). However, when [Mg(2+)] was decreased to 0.4 mM, SR Ca(2+) release occurred in 51% (16/33) and 6% (2/33) of MHN fibres after the addition of 1 mM halothane or 1 mM sevoflurane, respectively. Further decreases in [Mg(2+)] increased the proportion of responsive fibres. In the presence of 0.1 mM [Mg(2+)], Ca(2+) release occurred in all fibres (33/33) after the introduction of 1 mM halothane or 1 mM sevoflurane. In MHS fibres, 1 mM halothane or 1 mM sevoflurane-induced Ca(2+) release in 54% (7/13) or 15% (2/13) of fibres, respectively, at 1 mM Mg(2+). A decrease in [Mg(2+)] to 0.2 mM Mg(2+) was sufficient to render 100% of MHS fibres (13/13) responsive to 1 mM halothane or 1 mM sevoflurane. CONCLUSIONS: In both MHS and MHN fibres (i) halothane is a more potent activator of SR Ca(2+) release than sevoflurane and (ii) as with halothane, the efficacy of sevoflurane-induced SR Ca(2+) release exhibits a marked dependence on cytosolic [Mg(2+)]. The marked potentiation of SR Ca(2+) release after a moderate reduction in cytosolic [Mg(2+)] suggests that conditions which cause hypomagnesaemia will increase the probability and possibly severity of an MH event. Conversely, maintenance of a normal or slightly increased cytosolic [Mg(2+)] may reduce the probability of MH.

Metabolic factors contributing to altered Ca2+ regulation in skeletal muscle fatigue.

AIM: Skeletal muscle fatigue is characterized by a failure to maintain force production or power output during intense exercise. Many recent studies on isolated fibres have used brief repetitive tetanic contractions to mimic fatigue resulting from intensive exercise and to investigate the underlying cellular mechanisms. Such studies have shown that characteristic changes in Ca2+ regulation occur during fatiguing stimulation. This includes prolongation of the 'Ca2+-tails' which follow each period of tetanic stimulation and a progressive rise in resting [Ca2+]. More importantly, the final stage of fatigue is associated with a rapid decrease in tetanic [Ca2+]i and force. These fatigue-induced changes in sarcoplasmic reticulum (SR) Ca2+ regulation are temporally associated with alterations in the intracellular levels of phosphate metabolites and a causal relationship has often been proposed. The aim of this review is to evaluate the evidence linking changes in the levels of phosphate metabolites and altered Ca2+ regulation during fatigue. RESULTS: The following current hypotheses will be discussed: (1) the early changes in Ca2+ regulation reflect alterations in the intracellular levels of phosphate metabolites, (2) inhibition of the SR Ca2+ release mechanism (e.g. caused by ATP depletion and increased [Mg2+]) contributes to the decrease in tetanic [Ca2+]i during the final stages of fatigue and (iii) delayed entry of inorganic phosphate ions (Pi) into the SR, followed by precipitation of calcium phosphate (Ca-Pi), can explain the fatigue-induced decrease in tetanic [Ca2+]i. CONCLUSION: There is strong evidence that changes in phosphate metabolite levels contribute to early changes in SR Ca2+ regulation during fatigue and that inhibition of the SR Ca2+ release mechanism can partially explain the rapid decrease in tetanic [Ca2+]i during the final stages of fatigue. While precipitation of Ca-Pi may occur within the SR during fatigue, there is currently insufficient evidence to establish whether this contributes to the late decline in tetanic [Ca2+]i.

Mechanisms of reduced SR Ca(2+) release induced by inorganic phosphate in rat skeletal muscle fibers.

The effects of inorganic phosphate (P(i)) on Ca(2+) release from the sarcoplasmic reticulum (SR) were studied in mechanically skinned rat skeletal muscle fibers. Application of caffeine or T-tubule depolarization was used to induce Ca(2+) release from the SR, which was detected using fura 2 fluorescence. Addition of P(i) (1-40 mM) caused a reversible and concentration-dependent reduction in the caffeine-induced Ca(2+) transient. This effect was apparent at low P(i) concentration (<5 mM), which did not result in detectable precipitation of calcium phosphate within the SR. The inhibitory effect of P(i) exhibited a marked dependence on free Mg(2+) concentration. At 0.5 mM free Mg(2+), 5 mM P(i) reduced the caffeine-induced transient by 25.1 +/- 4.1% (n = 13). However, at 1.5 mM free Mg(2+), 5 mM P(i) reduced the amplitude of caffeine-induced Ca(2+) transients by 68.9 +/- 3.1% (n = 10). Depolarization-induced SR Ca(2+) release was similarly affected. These effects of P(i) may be important in skeletal muscle fatigue, if an inhibitory action of P(i) on SR Ca(2+) release is augmented by the rise in cytosolic Mg(2+) concentration, which accompanies ATP breakdown.

Effects of reactive oxygen species on aspects of excitation-contraction coupling in chemically skinned rabbit diaphragm muscle fibres.

Oxidants have been suggested to enhance contractile function in unfatigued muscle. In this study we aimed to determine the effect of oxidants on "chemically skinned" diaphragm muscle fibre bundles. The sarcoplasmic reticulum and contractile proteins were exposed to superoxide anions (O2-) and hydrogen peroxide (H2O2) under controlled conditions. Application of O2-initially increased maximum Ca2+ -activated force but subsequently reduced maximum Ca2+ -activated force without altering myofilament Ca2+ sensitivity. Unlike myocardium, caffeine-induced Ca2+ release from the sarcoplasmic reticulum was also inhibited by O2- exposure in diaphragm fibre bundles. Application of H2O2 also increased maximum Ca2+ -activated force but had additional effects on resting tension (which increased to 25 % of the control maximum Ca2+ -activated force). H2O2 was without effect on myofilament Ca2+ sensitivity or caffeine-induced Ca2+ release from the sarcoplasmic reticulum. These data demonstrate that oxidants can potentiate contractile force in the diaphragm through a direct action on the contractile proteins. The potentiation of force is not sustained, however, and under these conditions the detrimental effects of O2- on Ca2+ release from the sarcoplasmic reticulum combined with the effects of oxidants on the contractile proteins will ultimately compromise excitation-contraction coupling in the diaphragm. Experimental Physiology (2001) 86.2, 161-168.

Interdependent effects of inorganic phosphate and creatine phosphate on sarcoplasmic reticulum Ca2+ regulation in mechanically skinned rat skeletal muscle.

1. The effects of creatine phosphate (CP) and inorganic phosphate (Pi) on sarcoplasmic reticulum (SR) Ca2+ regulation were investigated in mechanically skinned muscle fibres from rat extensor digitorum longus (EDL) muscles. Changes in [Ca2+] were detected using fura-2 fluorescence, during continuous perfusion or when the solution surrounding the preparation was restricted to approximately 6 microl by stopping perfusion. 2. In solutions with 5 mM ATP and 10 mM CP, stopping the flow for 2-3 min had no effect on [Ca2+] within the bath. This suggests that SR Ca2+ uptake is balanced by an efflux under these conditions. 3. In solutions with CP, the introduction of Pi induced a small transient rise in [Ca2+], due to Ca2+ loss from the SR. Following equilibration with solutions containing Pi (> or = 5 mM), a maintained decrease in [Ca2+] occurred when the flow was stopped. This is consistent with calcium phosphate (Ca-Pi) precipitation within the SR, resulting in maintained Ca2+ uptake. 4. In the absence of CP, the [Ca2+] within the bath increased progressively when the flow was stopped. This rise in [Ca2+] was inhibited by an alternative ATP regenerating system comprising phosphoenolpyruvate (PEP) and pyruvate kinase (PK). Therefore, the loss of Ca2+ from the SR may result from local ADP accumulation and the consequent reversal of the SR Ca2+ pump. 5. In the absence of CP, the initial Ca2+ release associated with the introduction of Pi increased markedly. Following prolonged equilibration with solutions containing Pi, a rise in [Ca2+] occurred within the bath when the flow was stopped. Maintained Ca2+ uptake associated with Ca-Pi precipitation was not apparent at any level of Pi tested (1-60 mM), when CP was absent. 6. These results suggest that withdrawal of CP is associated with activation of a SR Ca2+ efflux pathway. This may involve reversal of the SR Ca2+ pump, due to local ADP accumulation. In the absence of CP, the dominant influence of Pi appears to involve further Ca2+ efflux via the SR Ca2+ pump. The possible relevance of these effects to skeletal muscle fatigue is considered.

Characteristics of phosphate-induced Ca(2+) efflux from the SR in mechanically skinned rat skeletal muscle fibers.

The effects of P(i) on sarcoplasmic reticulum (SR) Ca(2+) regulation were studied in mechanically skinned rat skeletal muscle fibers. Brief application of caffeine was used to assess the SR Ca(2+) content, and changes in concentration of Ca(2+) ([Ca(2+)]) within the cytosol were detected with fura 2 fluorescence. Introduction of P(i) (1-40 mM) induced a concentration-dependent Ca(2+) efflux from the SR. In solutions lacking creatine phosphate (CP), the amplitude of the P(i)-induced Ca(2+) transient approximately doubled. A similar potentiation of P(i)-induced Ca(2+) release occurred after inhibition of creatine kinase (CK) with 2,4-dinitrofluorobenzene. In the presence of ruthenium red or ryanodine, caffeine-induced Ca(2+) release was almost abolished, whereas P(i)-induced Ca(2+) release was unaffected. However, introduction of the SR Ca(2+) ATPase inhibitor cyclopiazonic acid effectively abolished P(i)-induced Ca(2+) release. These data suggest that P(i) induces Ca(2+) release from the SR by reversal of the SR Ca(2+) pump but not via the SR Ca(2+) channel under these conditions. If this occurs in intact skeletal muscle during fatigue, activation of a Ca(2+) efflux pathway by P(i) may contribute to the reported decrease in net Ca(2+) uptake and increase in resting [Ca(2+)].

Effects of creatine phosphate on Ca2+ regulation by the sarcoplasmic reticulum in mechanically skinned rat skeletal muscle fibres.

1. The effect of creatine phosphate (PCr) on sarcoplasmic reticulum (SR) Ca2+ regulation was studied in mechanically skinned skeletal muscle fibres from rat extensor digitorium longus (EDL). Preparations were perfused with solutions mimicking the intracellular milieu and the [Ca2+] within the muscle was monitored continuously using fura-2. 2. Brief application of 40 mM caffeine caused a transient increase in [Ca2+] due to SR Ca2+ release, and an associated tension response. Withdrawal of PCr resulted in (i) a slow transient release of Ca2+ from the SR (ii) a marked prolongation of the descending phase of the caffeine-induced fluorescence ratio transient and (iii) a decrease in the Ca2+ transient amplitude to 69.2 +/- 2.7 % (n = 16) of control responses. 3. Prolongation of the caffeine-induced Ca2+ transient also occurred following application of the SR Ca2+ pump inhibitor cyclopiazonic acid (CPA). This suggests that (i) the descending phase of the caffeine-induced Ca2+ transient is dependent on the rate of Ca2+ uptake by the SR and (ii) prolongation associated with PCr withdrawal may also reflect a decrease in the net Ca2+ uptake rate. 4. The effects of PCr withdrawal were mimicked by addition of the creatine kinase (CK) inhibitor 2,4-dinitro-1-fluorobenzene (DNFB). Hence, reducing the [PCr] may influence SR Ca2+ regulation by limiting local ATP regeneration by endogenous CK. After treatment with DNFB, PCr withdrawal had no effect on the Ca2+ transient, confirming that PCr does not have an additional direct effect on the SR. 5. The Ca2+ efflux associated with PCr withdrawal was insensitive to ryanodine or Ruthenium Red, but was effectively abolished by pretreatment with the SR Ca2+ pump inhibitor cyclopiazonic acid (CPA). This suggests that the Ca2+ efflux associated with PCr withdrawal is independent of the SR Ca2+ channel, but may involve reversal or inhibition of the Ca2+ ATPase. 6. These data suggest that Ca2+ regulation by the SR is strongly dependent on the supply of ATP via endogenous CK. Depletion of PCr may contribute to impaired SR Ca2+ regulation known to occur in intact skeletal muscle under conditions of fatigue.

Effects of caffeine and adenine nucleotides on Ca2+ release by the sarcoplasmic reticulum in saponin-permeabilized frog skeletal muscle fibres.

1. The effect of caffeine and adenine nucleotides on the sarcoplasmic reticulum (SR) Ca2+ release mechanism was investigated in permeabilized frog skeletal muscle fibres. Caffeine was rapidly applied and the resulting release of Ca2+ from the SR detected using fura-2 fluorescence. Decreasing the [ATP] from 5 to 0.1 mM reduced the caffeine-induced Ca2+ transient by 89 +/- 1.4% (mean +/- s.e.m., n = 16), while SR Ca2+ uptake was unaffected. 2. The dependence of caffeine-induced Ca2+ release on cytosolic [ATP] was used to study the relative ability of other structurally related compounds to substitute for, or compete with, ATP at the adenine nucleotide binding site. It was found that AMP, ADP and the non-hydrolysable analogue adenylyl imidodiphosphate (AMP-PNP) partially substituted for ATP, although none was as potent in facilitating the Ca2+-releasing action of caffeine. 3. Adenosine reversibly inhibited caffeine-induced Ca2+ release, without affecting SR Ca2+ uptake. Five millimolar adenosine markedly reduced the amplitude of the caffeine-induced Ca2+ transient by 64 +/- 4% (mean +/- s.e.m., n = 11). The degree of inhibition was dependent upon the cytosolic [ATP], suggesting that adenosine may act as a competitive antagonist at the adenine nucleotide binding site. 4. These data show that (i) the sensitivity of the in situ SR Ca2+ channel to caffeine activation is strongly dependent upon the cytosolic [ATP], (ii) the number of phosphates attached to the 5' carbon of the ribose ring influences the efficacy of the ligand, and (iii) removal of a single phosphate group transforms AMP from a partial agonist, to adenosine, which acts as a competitive antagonist under these conditions.

Effects of cyclopiazonic acid on Ca2+ regulation by the sarcoplasmic reticulum in saponin-permeabilized skeletal muscle fibres.

The effects of the sarcoplasmic reticulum (SR) Ca2+ pump inhibitor cyclopiazonic acid (CPA) were studied in saponin-permeabilized frog skeletal muscle fibres. Release of Ca2+ from the SR was triggered by brief (2 s) applications of 40 mM caffeine at 2-min intervals. Changes in [Ca2+] within the fibre were monitored continuously using Fura-2 fluorescence. At a bathing [Ca2+] of 100 nM, introduction of 20 microM CPA induced a slow release of Ca2+ from the SR. The following one to two caffeine-induced Ca2+ transients were markedly increased in amplitude and duration. Thereafter, the caffeine-induced Ca2+ transients decreased progressively and were barely detectable 6-7 min after introduction of CPA. However, increasing the bathing [Ca2+] or increasing the Ca2+ loading period resulted in a partial recovery of the caffeine-induced Ca2+ transients, suggesting that pump inhibition is incomplete, even in the presence of 100 microM CPA. The slow Ca2+ efflux induced by CPA was insensitive to ryanodine, but absent following abolition of SR Ca2+ pump activity by ATP withdrawal. These results suggest that the caffeine-induced Ca2+ transient reflects a balance between efflux via the SR Ca2+ channel and reuptake by the Ca pump. Ca2+ release upon addition of CPA may result from inhibition of SR Ca2+ uptake, which reveals a tonic Ca2+ efflux that is independent of the Ca2+ release channels.

The Wisconsin Age-related Macular Degeneration grading system: performance in an independent centre.

INTRODUCTION: Age-related Macular Degeneration (AMD) is the commonest cause of blindness in developed nations. Despite this, the epidemiology of AMD is poorly understood. A need for the documentation of AMD prevalence and incidence at a population level has stimulated the development of a comprehensive, observer-based, photographic grading method for AMD in Wisconsin. AIM: To independently assess the performance of the Wisconsin method by self-taught graders outside its centre of inception. METHOD: The inter-observer variability and confidence limits for detection of change were assessed for two self-taught graders (ophthalmologists). Self teaching was achieved exclusively from documentation and photographs provided by the system developers in Wisconsin. 295 retinal photographs of elderly people were independently assessed for 13 features by each of the two graders. RESULTS: Weighted and unweighted kappa statistics, % exact and one step apart agreement, and confidence limits for detection of change were calculated for the graded features on a "by eye' basis, and where appropriate, on a "by retinal subfield' basis. Levels of agreement for weighted kappa were moderate to substantial for most features. 95% and 90% confidence limits for significant change beyond measurement error were determined in terms of scale increments. CONCLUSION: We conclude that the Wisconsin AMD grading system can be independently learnt from documentation and photographs alone, and that an acceptable level of performance is attainable by self-taught graders.

Prevalence of age-related maculopathy at two points in time in an elderly British population.

UNLABELLED: As the demography of Western society changes, the population prevalence of diseases such as age-related macular degeneration (AMD) is expected to rise. Despite this, there remains a paucity of quality data concerning the population prevalence of AMD, the commonest cause of blindness in the elderly. PURPOSE: To report the prevalence of AMD at two points in time in an elderly population. METHOD: A geographically defined random population sample of elderly people was defined in 1980, and studied in 1982-4. In 1990, a cohort of survivors was identified. Participants underwent full ophthalmic examination with fundus photography using the same camera on each occasion. Photographs were randomly encoded and graded by two independent masked observers using the Wisconsin Age-related Maculopathy Grading System. Disagreements were resolved by consensus. RESULTS: Eighty-eight survivors participated in the follow-up examinations. Of these, 82 subjects had gradable retinal photographs for both examination points in at least one eye. There were 158 pairs of images (initial and subsequent) available for analysis. The mean age was 80 years (range 77-90 years) at the initial examination, and 87 years (range 84-97 years) at the subsequent examination; 70.7% of subjects were female. Prevalence rates for the initial examination were: drusen 72.8%, drusen confluence 37.3%, degeneration of the retinal pigment epithelium (RPE) 51.3%, increased pigment 22.2%, exudative AMD 1.9% and geographic atrophy 1.9%. Rates at second examination were: drusen 62.7% drusen confluence 41.8%, RPE degeneration 72.8%, increased pigment 16.5%, exudative AMD 3.8% and geographic atrophy 3.2%. CONCLUSION: This 'double' prevalence study provides detailed data on AMD lesions at two points in time in a population-based group of elderly people.

Seven year follow-up of age-related maculopathy in an elderly British population.

UNLABELLED: Despite age-related macular degeneration (AMD) being the commonest cause of blindness amongst the elderly in Western society, the incidence of new lesions is poorly documented and the natural history of existing disease remains ill understood. PURPOSE: To document in an elderly population the incidence of new AMD lesions and the progression of pre-existing AMD over time. METHOD: Baseline ophthalmic examinations were performed on a geographically defined random population sample of elderly people in 1982-4, and retinal photographs taken. The present study re-examined and re-photographed survivors after approximately 7 years using the same fundus camera. Photographs were randomly encoded, and independently graded for AMD features by two masked observers using the Wisconsin AMD grading system. Disagreements were resolved by review to reach a consensus. RESULTS: Eighty-two of the 88 participating survivors had photographs of gradable quality on both occasions in at least one eye. Mean age at follow-up was 87 years (range 84-97 years) and 70.7% of subjects were female. Paired photographs were available on 158 eyes, and showed important differences in drusen type, drusen area and characteristics of the retinal pigment epithelium (RPE) between initial and subsequent examinations. The 7 year incidence (and regression) of lesions was: drusen 30.6% (20.0%), RPE degeneration 54.5% (8.8%), increased pigment 11.6% (64.7%), subretinal haemorrhage 1.3%, subretinal scar/fibrin 1.3% and geographic study 1.3%. CONCLUSION: These unique population-based results provide new insight into the natural history of AMD in an elderly population.

Clinical manifestations of cows' milk allergy in childhood. II. The diagnostic value of skin tests and RAST.

In a study of cows' milk allergy (CMA) in infancy, 135 consecutive challenges were performed on children with a good clinical history of the disorder. Of these, only half of the patients were shown to have the disease. Highly atopic patients responded rapidly to small volumes of milk with acute urticaria, wheezing, stridor and eczema, whereas patients who were relatively non-atopic developed symptoms of eczema, bronchitis and wheezing over several hours or days. In a statistical evaluation of the diagnostic value of skin tests and RAST it was shown for the extracts used in this investigation, and for the population studied, all patients with SPT greater than or equal to 4 had CMA. The results highlight the potential diagnostic value of SPT in the identification of children with some forms of CMA if standardized cows' milk allergen extracts can be prepared.

A test for the assessment of disinfectant/sanitizers used for napkin storage.

A test is described for assessing the effect of hypochlorites in reducing the bacterial load on soiled napkins during storage, between removal from the infant and laundering.

Standards of cleansing and sterilization of infant feeding utensils in the home.

Microbiological standards achieved by mothers when sterilizing babies' feeding utensils in the home were studied in the Slough area in England. Results indicated a marked improvement in this aspect of baby hygiene when compared to results of a similar survey conducted in the town of Reading, England, in 1970. Previously, 78% of bottles and 70% of the teats were recorded as being satisfactorily sterilized. During this study the number of sterile bottles had risen to 98.1%, sterile teats to 90-6%. These improved standards may be an indication of better education of mothers.