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The Biochemical Journal Jun 1994We have investigated the modulation of stimulus-induced changes in intracellular Ca2+ concentration ([Ca2+]i) by a caffeine-and ryanodine-sensitive Ca2+ store in PC12...
We have investigated the modulation of stimulus-induced changes in intracellular Ca2+ concentration ([Ca2+]i) by a caffeine-and ryanodine-sensitive Ca2+ store in PC12 cells. In populations of fura-2-loaded cells, caffeine cause a concentration-dependent increase in [Ca2+]i that was saturable, reversible and inhibited in a use-dependent fashion by ryanodine. Maximal Ca2+ release occurred with 40 mM caffeine, with an EC50 of 13 mM caffeine and a Hill coefficient (h) of 2.7, indicating that the release mechanism was co-operative. Pretreatment of intact cell populations with increasing concentrations of caffeine in nominally Ca(2+)-free medium inhibited the subsequent Ca2+ response to a maximal concentration of ATP, in a dose-dependent manner. In permeabilized cells, a maximal concentration (40 microM) of InsP3 still released Ca2+ in the presence of a supramaximal concentration (50 mM) of caffeine, whereas caffeine was unable to release Ca2+ after the InsP3-sensitive store had been completely emptied. These data suggest that PC12 cells contain a uniquely InsP3-sensitive Ca2+ store, and a store that is sensitive to both InsP3 and caffeine. Depletion of the caffeine-sensitive Ca2+ store by caffeine and ryanodine pretreatment in intact cells attenuated the Ca2+ response to ATP, but not to 55 mM K+, suggesting that the caffeine-sensitive Ca2+ store acts as a Ca2+ source after ATP stimulation, but not after depolarization with 55 mM K+. Pretreatment of intact cells with ATP and ryanodine resulted in a use-dependent block of both caffeine- and ATP-mediated Ca2+ release, confirming that ATP stimulation of PC12 cells brings about activation of ryanodine receptors. The rate of recovery, but not the magnitude or rate of onset, of the depolarization-induced [Ca2+]i transient was modulated by the state of filling of the caffeine-sensitive Ca2+ store such that recovery was prolonged if the store was either full, or empty and unable to refill. We conclude that the caffeine- and ryanodine-sensitive Ca2+ store can act as a Ca2+ source and a Ca2+ sink in PC12 cells, and that its role may in part be governed by the nature of the stimulating agent.
Topics: Adenosine Triphosphate; Animals; Caffeine; Calcium; Calcium Channels; Inositol 1,4,5-Trisphosphate; Membrane Potentials; Muscle Proteins; PC12 Cells; Rats; Ryanodine; Ryanodine Receptor Calcium Release Channel; Theophylline
PubMed: 8002966
DOI: 10.1042/bj3000589 -
The Journal of Physiology Dec 2022The oligomeric form of the peptide amyloid beta 42 (Abeta42) contributes to the development of synaptic abnormalities and cognitive impairments associated with...
The oligomeric form of the peptide amyloid beta 42 (Abeta42) contributes to the development of synaptic abnormalities and cognitive impairments associated with Alzheimer's disease (AD). To date, there is a gap in knowledge regarding how Abeta42 alters the elementary parameters of GABAergic synaptic function. Here we found that Abeta42 increased the frequency and amplitude of miniature GABAergic currents as well as the amplitude of evoked inhibitory postsynaptic currents. When we focused on paired pulse depression (PPD) to establish whether GABA release probability was affected by Abeta42, we did not observe any significant change. On the other hand, a more detailed investigation of the presynaptic effects induced by Abeta42 by means of multiple probability fluctuation analysis and cumulative amplitude analysis showed an increase in both the size of the readily releasable pool responsible for synchronous release and the number of release sites. We further explored whether ryanodine receptors (RyRs) contributed to exacerbating these changes by stabilizing the interaction between RyRs and the accessory protein calstabin. We observed that the RyR-calstabin interaction stabilizer S107 restored the synaptic parameters to values comparable to those measured in control conditions. In conclusion, our results clarify the mechanisms of potentiation of GABAergic synapses induced by Abeta42. We further suggest that RyRs are involved in the control of synaptic activity during the early stage of AD onset and that their stabilization could represent a new therapeutical approach for AD treatment. KEY POINTS: Accumulation of the peptide amyloid beta 42 (Abeta42) is a key characteristic of Alzheimer's disease (AD) and causes synaptic dysfunctions. To date, the effects of Abeta42 accumulation on GABAergic synapses are poorly understood. The findings reported here suggest that, similarly to what is observed on glutamatergic synapses, Abeta42 modifies GABAergic synapses by targeting ryanodine receptors and causing calcium dysregulation. The GABAergic impairments can be restored by the ryanodine receptor-calstabin interaction stabilizer S107. Based on this research, RyRs stabilization may represent a novel pharmaceutical strategy for preventing or delaying AD.
Topics: Humans; Amyloid beta-Peptides; Ryanodine Receptor Calcium Release Channel; Ryanodine; Alzheimer Disease; Hippocampus; Neurons; Synapses; Synaptic Transmission
PubMed: 36284365
DOI: 10.1113/JP283537 -
The Journal of General Physiology Sep 1983To test whether ryanodine blocks the release of calcium from the sarcoplasmic reticulum in cardiac muscle, we examined its effects on the aftercontractions and transient...
To test whether ryanodine blocks the release of calcium from the sarcoplasmic reticulum in cardiac muscle, we examined its effects on the aftercontractions and transient depolarizations or transient inward currents developed by guinea pig papillary muscles and voltage-clamped calf cardiac Purkinje fibers in potassium-free solutions. Ryanodine (0.1-1.0 microM) abolished or prevented aftercontractions and transient depolarizations by the papillary muscles without affecting any of the other sequelae of potassium removal. In the presence of 4.7 mM potassium and at a stimulation rate of 1 Hz, ryanodine had only a small variable effect on papillary muscle force development and action potential characteristics. In calf Purkinje fibers, ryanodine (1 nM-1 microM) completely blocked the aftercontractions and transient inward currents without altering the steady state current-voltage relationship. Ryanodine also abolished the twitch in potassium-free solutions, but it enhanced the tonic force during depolarizing voltage-clamp steps. This latter effect was dependent on the combination of ryanodine and potassium-free solutions. The slow inward current was not blocked by 1 microM ryanodine, but ryanodine did appear to abolish an outward current that remained in the presence of 0.5 mM 4-aminopyridine. Our observations are consistent with the hypothesis that ryanodine, by inhibiting the release of calcium from the sarcoplasmic reticulum, prevents the oscillations in intracellular calcium that activate the transient inward currents and aftercontractions associated with calcium overload states.
Topics: Action Potentials; Alkaloids; Animals; Calcium; Cattle; Guinea Pigs; Male; Myocardial Contraction; Papillary Muscles; Purkinje Fibers; Ryanodine; Sarcoplasmic Reticulum; Solutions
PubMed: 6631403
DOI: 10.1085/jgp.82.3.385 -
The Journal of Biological Chemistry Nov 1987Ryanodine, a highly toxic alkaloid, reacts specifically with the Ca2+ release channels which are localized in the terminal cisternae of sarcoplasmic reticulum (SR). In...
Ryanodine, a highly toxic alkaloid, reacts specifically with the Ca2+ release channels which are localized in the terminal cisternae of sarcoplasmic reticulum (SR). In this study, the ryanodine receptor from cardiac SR has been purified, characterized, and compared with that of skeletal muscle SR. The ryanodine receptor was solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) in the presence of phospholipids. Purification was performed by sequential affinity chromatography followed by gel permeation chromatography in the presence of CHAPS and phospholipids. The enrichment of the receptor from cardiac microsomes was about 110-fold. The purified receptor contained a major polypeptide band of Mr 340,000 with a minor band of Mr 300,000 (absorbance ratio 100/8) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Electron microscopy of the purified receptor from heart showed square structures of 222 +/- 21 A/side, which is the unique characteristic of feet structures of junctional face membrane of terminal cisternae of SR. Recently, we isolated the ryanodine receptor from skeletal muscle (Inui, M., Saito, A., and Fleischer, S. (1987) J. Biol. Chem. 262, 1740-1747). The ryanodine receptors from heart and skeletal muscle have similar characteristics in terms of protein composition, morphology, chromatographic behavior, and Ca2+, salt, and phospholipid dependence of ryanodine binding. However, there are distinct differences: 1) the Mr of the receptor is slightly larger for skeletal muscle (Mr approximately 360,000); 2) the purified receptor from heart contains two different affinities for ryanodine binding with Kd values in the nanomolar and micromolar ranges, contrasting with that of skeletal muscle SR which shows only the high affinity binding; 3) the affinity of the purified cardiac receptor for ryanodine was 4-5-fold higher than that of skeletal muscle, measured under identical conditions. The greater sensitivity in ryanodine in intact heart can be directly explained by the tighter binding of the ryanodine receptor from heart. The present study suggests that basically similar machinery (the ryanodine receptor and foot structure) is involved in triggering Ca2+ release from cardiac and skeletal muscle SR, albeit there are distinct differences in the sensitivity to ryanodine and other ligands in heart versus skeletal muscle.
Topics: Animals; Cholic Acids; Chromatography, Gel; Dogs; Kinetics; Microsomes; Molecular Weight; Muscles; Myocardium; Receptors, Cholinergic; Ryanodine; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum
PubMed: 3680217
DOI: No ID Found -
The Journal of Physiology Sep 19951. The regulation of the cardiac Ca2+ release channel-ryanodine receptor (RyR) by exogenous acid phosphatase (AcPh) and purified Ca(2+)-calmodulin-dependent protein...
1. The regulation of the cardiac Ca2+ release channel-ryanodine receptor (RyR) by exogenous acid phosphatase (AcPh) and purified Ca(2+)-calmodulin-dependent protein kinase II (CaMKII) was studied in swine and rabbit sarcoplasmic reticulum (SR) vesicles using [3H]ryanodine binding and planar bilayer reconstitution experiments. 2. Addition of AcPh (1-20 U ml-1) to a standard incubation medium increased [3H]ryanodine binding in a Ca(2+)-dependent manner. Stimulation was only readily apparent in media containing micromolar Ca2+ concentrations. 3. Scatchard analysis of [3H]ryanodine binding curves revealed that AcPh enhanced binding by increasing the affinity of the receptor for [3H]ryanodine without recruiting additional receptor sites (Kd, 9.8 +/- 0.85 and 3.9 +/- 0.65 nM; Bmax (the maximal receptor density), 1.45 +/- 0.14 and 1.47 +/- 0.12 pmol mg-1 for control and AcPh, respectively). The failure of AcPh to increase Bmax suggested that the number of receptors that were 'dormant' due to phosphorylation in the SR preparation was very small. 4. At the single channel level, AcPh increased the open probability (Po) of RyR channels by increasing the opening rate and inducing the appearance of a longer open state while having no effect on single channel conductance. Thus AcPh acted directly on RyR channels or a closely associated regulatory protein. 5. CaMKII decreased both [3H]ryanodine binding and Po of RyRs when added to medium supplemented with micromolar levels of Ca2+ and calmodulin (CaM). Addition of a synthetic peptide inhibitor of CaMKII, or replacement of ATP with the non-hydrolysable ATP analogue adenylyl[beta, gamma-methylene]-diphosphate (AMP-PCP), prevented CaMKII inhibition of RyRs, suggesting that CaMKII acted specifically through a phosphorylation mechanism. 6. The inhibition of RyR channel activity by CaMKII was reversed by the addition of AcPh. Thus we showed that an in vitro phosphorylation-dephosphorylation mechanism effectively regulates RyRs. 7. The results suggest that intracellular signalling pathways that lead to activation of CaMKII may reduce efflux of Ca2+ from the SR by inhibition of RyR channel activity. The Ca2+ dependence of CaMKII inhibition suggests that the role of the phosphorylation mechanism is to modulate the RyR response to Ca2+.
Topics: Acid Phosphatase; Animals; Calcium Channels; Calcium-Calmodulin-Dependent Protein Kinases; In Vitro Techniques; Kinetics; Lipid Bilayers; Muscle Proteins; Myocardium; Phosphoprotein Phosphatases; Phosphorylation; Rabbits; Ryanodine; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum; Swine
PubMed: 8544125
DOI: 10.1113/jphysiol.1995.sp020904 -
Journal of Physiology and Pharmacology... Oct 2014Tetrabromobisphenol A (TBBPA) is a commonly used brominated flame retardant with recognized neuro- and cytotoxic properties that are presumably mediated by intracellular...
Tetrabromobisphenol A (TBBPA) is a commonly used brominated flame retardant with recognized neuro- and cytotoxic properties that are presumably mediated by intracellular Ca(2+) release. Other studies have demonstrated that ryanodine is able to inhibit Ca(2+) efflux from skeletal sarcoplasmic reticulum (SR) membranes in response to the known Ca(2+) releaser thapsigargin, provided that the macrocyclic brominated tyrosine derivative bastadin 5 is also present. Similar effects supporting the role of ryanodine receptors in thapsigargin-evoked Ca(2+) release have been observed in primary cultures of rat cerebellar granule cells (CGCs). Here, we used CGCs and the fluorescent intracellular Ca(2+) probe fluo-3 to test the following hypotheses: (1) TBBPA shares Ca(2+) releasing properties with thapsigargin, and (2) synthetic bastadin 12 can replace bastadin 5 as a pharmacological tool to identify these similarities. The results demonstrated that either 200 nM thapsigargin or 30 μM bastadin 12 alone induced an increase in the intracellular Ca(2+) level in CGCs, whereas 2.5 and 10 μM bastadin 12 had no effect on the basal Ca(2+) concentration. The thapsigargin-induced Ca(2+) release was partially reduced by co-administration of either 2.5 μM bastadin 12 or 200 μM ryanodine, and the release of Ca(2+) was nearly completely attenuated by these compounds when they were given together. TBBPA (5, 10 and 25 μM) administration caused a concentration-dependent increase in CGC Ca(2+) levels. Administration of 2.5 μM bastadin 12 with 200 μM ryanodine blocked the increase in intracellular Ca(2+) evoked by 10 μM TBBPA, although these compounds were ineffective when applied separately. These results indicate that bastadin 12 may replace bastadin 5 when testing the ability of ryanodine to inhibit Ca(2+) release from the intracellular stores of cultured neurons, and our findings support the hypothesis that TBBPA and thapsigargin induce intracellular Ca(2+) release through a common mechanism.
Topics: Animals; Calcium; Cells, Cultured; Cerebellum; Halogenated Diphenyl Ethers; Peptides, Cyclic; Polybrominated Biphenyls; Rats, Wistar; Ryanodine; Thapsigargin
PubMed: 25371527
DOI: No ID Found -
Modification of ryanodine toxicity by dantrolene and halothane in a model of malignant hyperthermia.Anesthesiology Sep 1980Ryanodine toxicity in animals has been suggested to constitute a model of malignant hyperthermia. Dantrolene is known to block the development of malignant hyperthermia...
Ryanodine toxicity in animals has been suggested to constitute a model of malignant hyperthermia. Dantrolene is known to block the development of malignant hyperthermia triggered by halothane in susceptible swine. The authors studied the influences of dantrolene and halothane on the effects of ryanodine in vitro in isolated rat diaphragm muscle segments, and in vivo in mice, to explore the validity of this model. In the diaphragm experiments, dantrolene was found to block or delay the development of contractures produced by ryanodine and to delay the potentiation of ryanodine-induced contractures caused by halothane. In mice, ryanodine at various dosages was injected and animals surviving after one hour were examined. Such survivors appeared grossly to be normal, and may constitute a model for the malignant hyperthermia patient. They were found to be susceptible to halothane and to succinylcholine, being killed by treatment with these two agents at dosages that were not lethal to control mice. Pretreatment of mice for 48 hours with orally administered dantrolene, followed by injection of ryanodine and then halothane anesthesia, decreased the lethality of ryanodine but did not reduce the number of deaths caused by the subsequent exposure to halothane. That the effects of ryanodine in vitro and in vivo are diminished and potentiated by dantrolene and halothane, respectively, would suggest that the ryanodine toxicity model of malignant hyperthermia may have validity and is worthy of further study. A prediction from this model is that the terminal cisternae of skeletal muscle sarcoplasmic reticulum may be altered in MH.
Topics: Alkaloids; Animals; Dantrolene; Diaphragm; Disease Models, Animal; Halothane; In Vitro Techniques; Male; Malignant Hyperthermia; Mice; Rats; Ryanodine; Succinylcholine
PubMed: 7425333
DOI: 10.1097/00000542-198009000-00004 -
The Journal of Biological Chemistry May 2003Maurocalcine (MCa) isolated from Scorpio maurus palmatus venom shares 82% sequence identity with imperatoxin A. Both scorpion toxins are putative mimics of the II-III...
Maurocalcine (MCa) isolated from Scorpio maurus palmatus venom shares 82% sequence identity with imperatoxin A. Both scorpion toxins are putative mimics of the II-III loop peptide (termed peptide A (pA)) of alpha(1s)-dihydropyridine receptor and are thought to act at a common site on ryanodine receptor type 1 (RyR1) important for skeletal muscle EC coupling. The relationship between the actions of synthetic MCa (sMCa) and pA on RyR1 were examined. sMCa released Ca(2+) from SR vesicles (EC(50) = 17.5 nm) in a manner inhibited by micromolar ryanodine or ruthenium red. pA (0.5-40 microm) failed to induce SR Ca(2+) release. Rather, pA enhanced Ca(2+) loading into SR and fully inhibited Ca(2+)-, caffeine-, and sMCa-induced Ca(2+) release. The two peptides modified single channel gating behavior in distinct ways. With Cs(+)-carrying current, 10 nm to 1 microm sMCa induced long lived subconductances having 48% of the characteristic full open state and occasional transitions to 29% at either positive or negative holding potentials. In contrast, pA stabilized long lived channel closures with occasional burst transitions to 65% (s1) and 86% (s2) of the full conductance. The actions of pA and sMCa were observed in tandem. sMCa stabilized additional subconductance states proportional to pA-induced subconductances (i.e. 43% of pA-modified s1 and s2 substates), revealing a proportional gating mechanism. [(3)H]Ryanodine binding and surface plasmon resonance analyses indicated that the peptides did not interact by simple competition for a single class of mutually exclusive sites on RyR1 to produce proportional gating. The actions of sMCa were also observed with ryanodine-modified channels and channels deficient in immunophilin 12-kDa FK506-binding protein. These results provide evidence that sMCa and pA stabilize distinct RyR1 channel states through distinct mechanisms that allosterically stabilize gating states having proportional conductance.
Topics: Amino Acid Sequence; Animals; Caffeine; Calcium; Ion Channel Gating; Molecular Sequence Data; Oncogene Protein pp60(v-src); Peptide Fragments; Rabbits; Ryanodine; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum; Scorpion Venoms
PubMed: 12586831
DOI: 10.1074/jbc.M209501200 -
The Journal of Biological Chemistry Nov 1981Two subpopulations of cardiac sarcoplasmic reticulum vesicles were resolved functionally, based on their sensitivities to the drug ryanodine. These two subpopulations of...
Two subpopulations of cardiac sarcoplasmic reticulum vesicles were resolved functionally, based on their sensitivities to the drug ryanodine. These two subpopulations of sarcoplasmic reticulum vesicles, termed ryanodine-sensitive and ryanodine-insensitive, were separated by preloading crude cardiac microsomes with Ca2+ oxalate in the presence of ATP, followed by sucrose density gradient centrifugation. Ryanodine-insensitive vesicles accumulated most of the Ca2+ oxalate during the preload, and constituted the densest subfraction recovered from the sucrose gradient. These ryanodine-insensitive vesicles exhibited the highest density of Ca2+ pumps, and accounted for 10 to 15% of the total protein in crude cardiac microsomes. Ryanodine-insensitive vesicles continued to transport substantial amounts of Ca2+ after isolation. Ryanodine-sensitive vesicles accumulated negligible Ca2+ during the preload, and were recovered from the lower density regions of the sucrose gradient. On a milligrams of protein basis, these vesicles were present in 7-fold excess over ryanodine-insensitive vesicles. Ryanodine-sensitive vesicles transported low amounts of Ca2+ under normal incubation conditions, but 3 X 10(-4) M ryanodine strikingly increased their Ca2+ uptake 5- to 10-fold. Ca2+ uptake by ryanodine-sensitive vesicles was uniquely regulated by Ca2+ ion concentration. Elevation of the ionized Ca2+ concentration from 2 to 4 microM increased Ca2+ uptake by these vesicles greater than 5-fold, but had no effect on their Ca2+-dependent ATPase activity. These ryanodine- and Ca2+ concentration-dependent effects were apparent for only ryanodine-sensitive vesicles. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed distinct differences in polypeptide staining between ryanodine-sensitive and ryanodine-insensitive vesicles, confirming by an independent method that the two populations of vesicles were different. These data provide the first biochemical evidence for functional and structural heterogeneity of cardiac sarcoplasmic reticulum vesicles.
Topics: Adenosine Triphosphate; Animals; Biological Transport, Active; Calcium; Calcium-Transporting ATPases; Heart Ventricles; Kinetics; Myocardium; Ryanodine; Sarcoplasmic Reticulum; Sodium-Potassium-Exchanging ATPase
PubMed: 6271762
DOI: No ID Found -
Yonsei Medical Journal Dec 1993Ryanodine has different effects on the contractility of rat and guinea pig ventricular muscle. Thus we investigated the effect of ryanodine on the intracellular Ca2+ and...
Ryanodine has different effects on the contractility of rat and guinea pig ventricular muscle. Thus we investigated the effect of ryanodine on the intracellular Ca2+ and Na+ activities of the rat and guinea pig ventricular myocytes with two specific aims; whether there are any differences in intracellular Na+ activities between rat and guinea pig ventricular muscle cells, and if any, how the differences in intracellular Na+ activities are related to the effect of Na(+)-Ca2+ exchange on the action potential configuration and excitation-contraction coupling of the rat and guinea pig ventricular myocytes. Ryanodine (10(-7) M) diminished the slow repolarization phase of the rat ventricular action potential while the duration of the rapid repolarization phase increased. Ryanodine (10(-7) M) significantly increased the plateau of the action potential. At the steady state of 0.2 cps, intracellular Na+ activities (aiNa) of the rat and guinea pig ventricular myocytes were 8.7 +/- 5.2 mM (n = 16, 4 rats) and 10.0 +/- 4.1 mM (n = 25, 7 guinea pigs) respectively, but there were no statistically significant differences. The contractility of the rat ventricular muscle nearly disappeared due to ryanodine (10(-7) M) with little changes in aiNa. Monensin (10 mM) not only increased the resting tension but also remarkably increased aiNa from 2.0 mM to 20 mM. Ryanodine (10(-7) M) continuously decreased aiNa of the guinea pig ventricular muscle after the contraction ceased to decrease. Monensin increased the contractility as well as aiNa. These results suggest that the contractility of rat and guinea pig ventricular myocytes is determined by the change in the action of the Na(+)-Ca2+ exchange mechanism depending upon the plateau of action potential and the intracellular Na+ and Ca2+ activities. So ryanodine could decreases the contractility via its effect on Na(+)-Ca2+ exchange transport which could be one of possible mechanisms of negative inotropism by ryanodine.
Topics: Action Potentials; Animals; Female; Guinea Pigs; Heart; Heart Ventricles; Intracellular Membranes; Male; Myocardial Contraction; Myocardium; Rats; Ryanodine; Sodium
PubMed: 8128735
DOI: 10.3349/ymj.1993.34.4.311