Assessing the pathogenicity of RYR1 variants in malignant hyperthermia.

BACKGROUND: . Missense variants in the ryanodine receptor 1 gene ( RYR1 ) are associated with malignant hyperthermia but only a minority of these have met the criteria for use in predictive DNA diagnosis. We examined the utility of a simplified method of segregation analysis and a functional assay for determining the pathogenicity of recurrent RYR1 variants associated with malignant hyperthermia. METHODS: . We identified previously uncharacterised RYR1 variants found in four or more malignant hyperthermia families and conducted simplified segregation analyses. An efficient cloning and mutagenesis strategy was used to express ryanodine receptor protein containing one of six RYR1 variants in HEK293 cells. Caffeine-induced calcium release, measured using a fluorescent calcium indicator, was compared in cells expressing each variant to that in cells expressing wild type ryanodine receptor protein. RESULTS.: We identified 43 malignant hyperthermia families carrying one of the six RYR1 variants. There was segregation of genotype with the malignant hyperthermia susceptibility phenotype in families carrying the p.E3104K and p.D3986E variants, but the number of informative meioses limited the statistical significance of the associations. HEK293 functional assays demonstrated an increased sensitivity of RyR1 channels containing the p.R2336H, p.R2355W, p.E3104K, p.G3990V and p.V4849I compared with wild type, but cells expressing p.D3986E had a similar caffeine sensitivity to cells expressing wild type RyR1. CONCLUSIONS: . Segregation analysis is of limited value in assessing pathogenicity of RYR1 variants in malignant hyperthermia. Functional analyses in HEK293 cells provided evidence to support the use of p.R2336H, p.R2355W, p.E3104K, p.G3990V and p.V4849I for diagnostic purposes but not p.D3986E.

Diverse mechanisms underlying the regulation of ion channels by carbon monoxide.

Carbon monoxide (CO) is firmly established as an important, physiological signalling molecule as well as a potent toxin. Through its ability to bind metal-containing proteins, it is known to interfere with a number of intracellular signalling pathways, and such actions can account for its physiological and pathological effects. In particular, CO can modulate the intracellular production of reactive oxygen species, NO and cGMP levels, as well as regulate MAPK signalling. In this review, we consider ion channels as more recently discovered effectors of CO signalling. CO is now known to regulate a growing number of different ion channel types, and detailed studies of the underlying mechanisms of action are revealing unexpected findings. For example, there are clear areas of contention surrounding its ability to increase the activity of high conductance, Ca(2+) -sensitive K(+) channels. More recent studies have revealed the ability of CO to inhibit T-type Ca(2+) channels and have unveiled a novel signalling pathway underlying tonic regulation of this channel. It is clear that the investigation of ion channels as effectors of CO signalling is in its infancy, and much more work is required to fully understand both the physiological and the toxic actions of this gas. Only then can its emerging use as a therapeutic tool be fully and safely exploited.

Genetic variation in RYR1 and malignant hyperthermia phenotypes.

BACKGROUND: Malignant hyperthermia (MH) is associated, in the majority of cases, with mutations in RYR1, the gene encoding the skeletal muscle ryanodine receptor. Our primary aim was to assess whether different RYR1 variants are associated with quantitative differences in MH phenotype. METHODS: The degree of in vitro pharmacological muscle contracture response and the baseline serum creatine kinase (CK) concentration were used to generate a series of quantitative phenotypes for MH. We then undertook the most extensive RYR1 genotype-phenotype correlation in MH to date using 504 individuals from 204 MH families and 23 RYR1 variants. We also determined the association between a clinical phenotype and both the laboratory phenotype and RYR1 genotype. RESULTS: We report a novel correlation between the degree of in vitro pharmacological muscle contracture responses and the onset time of the clinical MH response in index cases (P<0.05). There was also a significant correlation between baseline CK concentration and clinical onset time (P=0.039). The specific RYR1 variant was a significant determinant of the severity of each laboratory phenotype (P<0.0001). CONCLUSIONS: The MH phenotype differs significantly with different RYR1 variants. Variants leading to more severe MH phenotype are distributed throughout the gene and tend to lie at relatively conserved sites in the protein. Differences in phenotype severity between RYR1 variants may explain the variability in clinical penetrance of MH during anaesthesia and why some variants have been associated with exercise-induced rhabdomyolysis and heat stroke. They may also inform a mutation screening strategy in cases of idiopathic hyperCKaemia.

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.

Na+-Ca2+ exchange activity is localized in the T-tubules of rat ventricular myocytes.

Detubulation of rat ventricular myocytes has been used to investigate the role of the t-tubules in Ca2+ cycling during excitation-contraction coupling in rat ventricular myocytes. Ca2+ was monitored using fluo-3 and confocal microscopy. In control myocytes, electrical stimulation caused a spatially uniform increase in intracellular [Ca2+] across the cell width. After detubulation, [Ca2+] rose initially at the cell periphery and then propagated into the center of the cell. Application of caffeine to control myocytes resulted in a rapid and uniform increase of intracellular [Ca2+]; the distribution and amplitude of this increase was the same in detubulated myocytes, although its decline was slower. On application of caffeine to control cells, there was a large, rapid, and transient rise in extracellular [Ca2+] as Ca2+ was extruded from the cell; this rise was significantly smaller in detubulated cells, and the remaining increase was blocked by the sarcolemmal Ca2+ ATPase inhibitor carboxyeosin. The treatment used to produce detubulation had no significant effect on Ca2+ efflux in atrial cells, which lack t-tubules. Detubulation of ventricular myocytes also resulted in loss of Na+-Ca2+ exchange current, although the density of the fast Na+ current was unaltered. It is concluded that Na+-Ca2+ exchange function, and hence Ca2+ efflux by this mechanism, is concentrated in the t-tubules, and that the concentration of Ca2+ flux pathways in the t-tubules is important in producing a uniform increase in intracellular Ca2+ on stimulation.

Effects of cytosolic ATP on Ca(2+) sparks and SR Ca(2+) content in permeabilized cardiac myocytes.

Confocal imaging was used to study the influence of cytosolic ATP on the properties of spontaneous Ca(2+) sparks in permeabilized ventricular myocytes. Cells were perfused with mock intracellular solutions containing fluo 3. Reducing [ATP] to <0.5 mmol/L decreased the frequency but increased the amplitude of spontaneous Ca(2+) sparks. In the presence of 20 micromol/L ATP, the amplitude increased by 48.7+/-10.9%, and the frequency decreased by 77.07+/-3.8%, relative to control responses obtained at 5 mmol/L ATP. After exposure to a solution containing zero ATP, the frequency of Ca(2+) sparks decreased progressively and approached zero within 90 seconds. As ATP washed out of the cell, the sarcoplasmic reticulum (SR) Ca(2+) content increased, until reaching a maximum after 3 minutes. Subsequent introduction of adenylyl imidodiphosphate precipitated a burst of large-amplitude Ca(2+) sparks. This was accompanied by a rapid decrease in SR Ca(2+) content to 80% to 90% of the steady-state value obtained in the presence of 5 mmol/L ATP. Thereafter, the SR Ca(2+) content declined much more slowly over 5 to 10 minutes. The effects of ATP withdrawal on Ca(2+) sparks may reflect reduced occupancy of the adenine nucleotide site on the SR Ca(2+) channel. These effects may contribute to previously reported changes in SR function during myocardial ischemia and reperfusion, in which ATP depletion and Ca(2+) overload occur.

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.

Effects of cytosolic ATP on spontaneous and triggered Ca2+-induced Ca2+ release in permeabilised rat ventricular myocytes.

1. The effects of cytosolic ATP on sarcoplasmic reticulum (SR) Ca2+ regulation were investigated in saponin-permeabilised rat ventricular myocytes. [Ca2+] within the cells was monitored using Fura-2 or Fluo-3 fluorescence. Spontaneous cyclic Ca2+ release from the SR was induced by increasing the bathing [Ca2+] to 200-300 nM, in solutions weakly Ca2+ buffered with 0.05 mM EGTA. Alternatively, Ca2+-induced Ca2+ release (CICR) was triggered by a rapid increase in [Ca2+] induced by flash photolysis of Nitr-5 (0.08 mM), replacing EGTA in the solution. 2. Stepwise reductions in [ATP] were associated with corresponding decreases in the frequency and increases in the amplitude of spontaneous Ca2+ transients. A decrease from 5 mM to 0. 1 mM ATP, reduced the release frequency by 48.6 +/- 7 % (n = 7) and almost doubled the amplitude of the Ca2+ transient. Marked prolongation of the spontaneous Ca2+ transient occurred when [ATP] was further reduced to 10 microM, consistent with inhibition of the SR Ca2+ pump. 3. These effects of ATP were compared with other interventions that inhibit Ca2+ uptake or reduce the sensitivity of the SR Ca2+ release mechanism. Inhibition of the SR Ca2+ pump with cyclopiazonic acid (CPA) markedly reduced the spontaneous Ca2+ release frequency, without changing the amplitude. The descending phase of the Ca2+ transient was prolonged in the presence of CPA, while the rising phase was unaffected. In contrast, desensitisation of the SR Ca2+ release mechanism with tetracaine decreased the frequency of spontaneous release, but markedly increased the amplitude. 4. CICR triggered by flash photolysis of Nitr-5 appeared to be more sensitive to cytosolic [ATP] than spontaneous release and was generally delayed by a decrease to 2.5 mM ATP. In the presence of 0.1-0.2 mM ATP, release often failed completely or was not consistently triggered. Some preparations exhibited Ca2+ release 'alternans', whereby every alternate trigger induced a response. 5. These results suggest that the increase in spontaneous Ca2+ release amplitude and the decrease in frequency that occurs as [ATP] is reduced from 1 mM to 100 microM, is mainly due to desensitisation of the SR Ca2+ release mechanism, which allows the SR Ca2+ content to reach a higher level before release occurs. At very low [ATP], a reduction in the SR Ca2+ uptake rate may also contribute to the decrease in release frequency. CICR triggered by photolysis of Nitr-5 appeared to be more sensitive to cytosolic [ATP]. The possible underlying mechanisms and the relevance of these results to myocardial ischaemia or hypoxia 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.

Measurement of SR Ca2+ content in the presence of caffeine in permeabilised rat cardiac trabeculae.

This study was designed to measure the Ca2+ content of rat cardiac sarcoplasmic reticulum (SR) after equilibration with normal diastolic levels of Ca2+ (100 nM), in the absence and presence of caffeine. Measurements of [Ca2+] based on Fura-2 fluorescence were made from a limited bath volume (230 nl) containing individual saponin-permeabilised rat cardiac trabeculae. Injection of caffeine (5-40 mM) into this volume caused an initial release of Ca2+ from the SR, but within 30 s the SR was able to re-accumulate a significant proportion of the Ca2+. Ca2+ re-accumulation into the SR could be prevented by removal of ATP to inhibit the SR Ca2+ pump. Incubation of the preparation in an ATP-containing solution containing caffeine (5-40 mM) and 100 nM Ca2+ indicated that the SR's ability to retain Ca2+ depends inversely on the dose of caffeine. The relative Ca2+ content of the SR after preincubation with caffeine was 86.7+/-3.5% at a caffeine concentration of 5 mM, 62.5+/-5.1% at 10 mM caffeine, 37.8+/-8.1% at 20 mM caffeine and 7. 1+/-1.9% at 40 mM caffeine. Measurement of the SR Ca2+ release in the presence of different BAPTA concentrations was used to calculate (1) the Ca2+-binding capacity of the preparation (equivalent to 245+/-10 microM BAPTA) and (2) the Ca2+ content of the SR accessed by caffeine after equilibration with 100 nM Ca2+ (186+/-11 micromol/l cell volume or 5.6 mmol/l SR volume).

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.

Effects of cytosolic Ca2+ on the Ca2+ content of the sarcoplasmic reticulum in saponin-permeabilized rat ventricular trabeculae.

Rat ventricular trabeculae were mounted for isometric tension recording, and then permeabilized with saponin. The Ca2+ concentration ([Ca2+]) within the permeabilized preparation (cytosolic [Ca2+]) was monitored continuously using Indo-1 and the integrals of Ca2+ transients resulting from brief caffeine application used as an index of the sarcoplasmic reticulum (SR) Ca2+ content. The relationship between SR Ca2+ content and cytosolic [Ca2+] was studied within the reported physiological range (i.e. 50-250 nmol . l-1 Ca2+). Increasing cytosolic [Ca2+] from 50 nmol . l-1 to 250 nmol . l-1 increased the steady-state SR Ca2+ content about threefold. However, increasing [Ca2+] above 250 nmol . l-1 typically resulted in spontaneous SR Ca2+ release, with no further increase in SR Ca2+ content. The SR Ca2+ content increased only slowly when cytosolic [Ca2+] was increased; it was unchanged 20 s after a rapid increase in cytosolic [Ca2+], but increased progressively to a new steady-state level during the following 1-2 min. In a parallel series of experiments using intact papillary muscles, increasing extracellular [Ca2+] (from 0.5 to 5 mmol . l-1) significantly increased twitch tension within 20 s of the solution change. These results support previous suggestions that the SR Ca2+ content may increase when diastolic cytosolic [Ca2+] rises during inotropic interventions such as increased stimulus rate or extracellular [Ca2+]. However, the rate at which SR Ca2+ responds to changes in cytoplasmic [Ca2+] within the diastolic range does not appear rapid enough to explain the early potentiation of twitch tension in intact preparations after an increase in extracellular [Ca2+].

Comparative effects of inorganic phosphate and oxalate on uptake and release of Ca2+ by the sarcoplasmic reticulum in saponin skinned rat cardiac trabeculae.

1. Ventricular trabeculae from the right ventricle of rat heart were suspended in a 6 microliters bath and "skinned' with saponin (50 mg ml-1). Preparations were perfused with solutions mimicking the intracellular milieu and the [Ca2+] within the bath was monitored continuously using fura-2. 2. Application of 20 mM caffeine released Ca2+ from the sarcoplasmic reticulum (SR), resulting in a transient increase in the fura-2 fluorescence ratio. Caffeine-induced Ca2+ transients were smaller in the presence of 30 or 60 mM inorganic phosphate (Pi). This depressive effect of Pi on SR function was reversed by 10 mM creatine phosphate (CP). Caffeine-induced Ca2+ transients were also reduced in the presence of 10 mM oxalate, although this effect was not reversed by CP. 3. When perfusion was stopped in the presence of 30 or 60 mM Pi, the [Ca2+] within the bath remained constant. However, when the flow was stopped in the presence of 60 mM Pi and 10 mM CP, a prolonged decrease in [Ca2+] occurred, consistent with precipitation of calcium phosphate within the SR. A similar decrease in [Ca2+] was observed when perfusion was stopped in the presence of 2 or 20 mM oxalate, in the absence or presence of CP. 4. The SR was Ca2+ depleted by withdrawal of ATP and exposure to 20 mM caffeine. Perfusion was then stopped and ATP reapplied, resulting in a maintained decrease in [Ca2+] within the bath, due to SR Ca2+ uptake. Net Ca2+ uptake was markedly reduced in the presence of 30 mM Pi. In contrast, 20 mM oxalate increased Ca2+ uptake and the [Ca2+] within the bath continued to fall over 2-3 min. 5. Introduction of Pi released Ca2+ from the SR. Ryanodine (100 microM) abolished caffeine-induced Ca2+ release while Pi-induced Ca2+ release was unaffected. Pi-induced Ca2+ release was reduced in the constant presence of 20 mM caffeine or 10 mM CP and was abolished completely by disruption of the SR membrane with Triton X-100. Pi-induced Ca2+ release occurred after abolition of SR Ca2+ uptake by ATP withdrawal. 6. These results suggest that the Pi-induced decrease in releasable Ca2+ does not result from precipitation of calcium phosphate within the SR lumen. Pi inhibits net SR Ca2+ uptake, but this appears to result from activation of a ryanodine-insensitive Ca2+ efflux pathway rather then inhibition of Ca2+ uptake. Possible mechanisms are considered, including reversal of the SR Ca2+ pump.

Effects of creatine phosphate and inorganic phosphate on the sarcoplasmic reticulum of saponin-treated rat heart.

1. Ventricular trabeculae from rat heart were permeabilized by treatment with saponin. In the presence of 150 nM Ca2+, application of 20 mM caffeine released Ca2+ from the sarcoplasmic reticulum (SR), resulting in a transient contracture. Ca2+ released from the SR was detected using fura-2 fluorescence. The amplitudes of the caffeine-induced Ca2+ transients were used to assess SR Ca2+ content. 2. In the absence of creatine phosphate (CP), introduction of 5-30 mM inorganic phosphate (Pi) caused a net release of Ca2+ from the SR. Subsequent caffeine-induced Ca2+ and tension transients were smaller in the presence of Pi. Under these conditions, 30 mM Pi decreased the caffeine-induced Ca2+ transients by 45 +/- 3.1% (mean +/- S.D., n = 14). On removal of Pi, the [Ca2+] transiently decreased and the caffeine-induced Ca2+ transients returned to control levels over 4-6 min. 3. In the presence of CP (5-15 mM), the Ca2+ transients were unaffected by the introduction of Pi (5-30 mM) or slightly increased in amplitude. Pi (30 mM) significantly increased the caffeine-induced Ca2+ transients by 7 +/- 8.8% (mean +/- S.D., n = 19, P < 0.05) in the presence of 15 mM CP. The release of Ca2+ on addition of Pi and decrease in [Ca2+] on Pi withdrawal was less pronounced or absent completely in the presence of CP. The inhibitory effects of Pi on caffeine-induced Ca2+ release became apparent as the [CP] was decreased from 5 to 0 mM. 4. In the presence of the creatine phosphokinase inhibitor dinitro-fluorobenzene (DNFB) the effects of Pi (in the presence of CP) were qualitatively similar to the results obtained in the absence of CP, although the decrease in caffeine-induced Ca2+ release was less pronounced. 5. These results suggest that the rise in [Pi]i during ischaemia or anoxia will have little effect on the regulation of Ca2+ by the SR while the [CP]i remains above 5 mM. However, as the [CP] decreases below 5 mM, the accumulation of Pi within the cytosol will progressively reduce the SR Ca2+ content. CP may act in conjunction with endogenous creatine phosphokinase to modify the response of the SR to Pi, and possible mechanisms are considered.