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PubMed2002

Results of contracture tests with halothane, caffeine, and ryanodine depend on different malignant hyperthermia-associated ryanodine receptor gene mutations.

BACKGROUND: More than 20 mutations in the gene encoding for the ryanodine receptor (RYR1), a Ca2+ release channel of the skeletal muscle sarcoplasmic reticulum, have been found to be associated with malignant hyperthermia (MH). This study was designed to investigate the effects of different mutations in the RYR1 gene on contracture development in in vitro contracture tests (IVCT) with halothane, caffeine, and ryanodine. METHODS: Ninety-three MH-susceptible (MHS) patients, diagnosed by the standard IVCT with halothane and caffeine, were included in this prospective study. Surplus muscle specimens were used for an IVCT with 1 microm ryanodine. The contracture course during the ryanodine IVCT was described by the attainment of different time points: onset time of contracture and times when contracture reached 2 mN or 10 mN. In addition, all patients were screened for mutations of the RYR1 gene. RESULTS: In 36 patients, four different mutations of the RYR1 gene (C487-T, G1021-A, C1840-T, G7300-A) were found. The IVCT threshold concentrations of halothane and caffeine were lower in patients with the C487-T mutation compared with patients without a detected mutation in the RYR1 gene. In the IVCT with ryanodine, contracture levels of 2 mN and 10 mN were reached earlier in muscle specimens from patients with C487-T, C1840-T, and G7300-A mutations compared with specimens from patients with the G1021-A mutation and patients without detected mutation in the RYR1 gene. CONCLUSIONS: The differences between the groups in the halothane and caffeine IVCT threshold concentrations and in the time course of contracture development in the ryanodine IVCT underline the hypothesis that certain mutations in the RYR1 gene could make the ryanodine receptor more sensitive to specific ligands. This may be an explanation for varying clinical symptoms of MH crisis in humans.

Adolescent↗
PubMed1997

Conformation state of the ryanodine receptor and functional effects of ryanodine on skeletal muscle.

The ryanodine receptor (RyR) and the dihydropyridine (DHP) receptor (L-channels) comprise the main elements of the functional feet of the triadic element in skeletal muscle. These two main elements have conformational states that are regulated by the membrane potential and the consequent electrical field. The pharmacological action of ryanodine on skeletal muscle depends upon the physiological functional state of the RyR. At a resting potential of -90 m V, ryanodine at very low concentrations, 10(-11) M, causes the RyR to have a low conductance state which allows calcium to leak from the terminal cisternae of the sarcoplasmic reticulum and to be recycled with ATP utilization, leading to a marked increase in oxygen consumption and aerobic metabolism. At concentrations greater than 10(-6) M, ryanodine can cause a slowly developing contracture of resting muscle, inhibit the muscle twitch when the RyR complex is formed during stimulation, and, if formed before stimulation, accelerate the development of contracture. Biochemical studies have revealed that the RyR has four binding sites in which the conductance state depends upon the number of sites occupied by ryanodine. Our present understanding of the RyR-operated calcium channel is the result of an interdisciplinary approach in which each discipline (anatomy, physiology, biophysics, and biochemistry) contributes to our knowledge of the pharmacological action of ryanodine.

Animals↗
PubMed2000

Pharmacological clues to calmodulin-mediated activation of skeletal ryanodine receptor using [3H]-ryanodine binding.

The hypothesis that calmodulin (CaM) may act as a positive modulator of junctional SR Ca2+-release channel/ ryanodine receptor (RyRl) rests largerly on the demonstrated capacity of CaM to interact structurally and functionally with RyRl at pCa > 8 (Tripathy et al., 1995). The goal of the present [3H]-ryanodine binding study was to produce, in isolated terminal cisternae (TC) and in purified junctional face membrane (JFM), CaM-mediated activation of RyRl at less extreme pCa values, i.e. closer to resting myoplasmic pCa, and to analyze more accurately the corresponding changes in binding affinity for ryanodine of the receptor. We were able to monitor these changes at an optimum pCa of 6.5, following pre-activation of native RyRl by mM concentrations of caffeine or microM concentrations of antraquinone compound doxorubicin, and at various doses of these triggers. CaM increased the affinity of ryanodine binding to isolated TC in the presence of 1 mM AMP-PCP as an activator of RyRl; the Kd for ryanodine binding was reduced from 21.8 nM to 13.2 nM by 1microM CaM. Similar effects of CaM were seen when AMP-PCP was replaced by either caffeine or doxorubicin. In order to discount the involvement of SR extrajunctional proteins in this effect, the experiments were repeated on purified JFM. Again, CaM increased the affinity of ryanodine binding; the Kd was reduced from 11.1 nM to 7.0 nM by 1 microM CaM (in the presence of doxorubicin). Pharmacological triggers of CaM-activatory action on native RyRl, like caffeine and doxorubicin, have long been characterized for their ability to activate RyRl by increasing the Ca2+-sensitivity of the receptor. We speculate that the triggering effect of these agents on the CaM-mediated mechanism in vitro might mimick one of the early effects of the activation of RyRl in skeletal muscle, during E-C coupling.

Adenosine Triphosphate↗
PubMed1994

Ryanodine and dihydropyridine binding patterns and ryanodine receptor mRNA levels in myopathic hamster heart.

We have determined the densities of sarcolemmal voltage-dependent Ca2+ channels (VDCC) and Ca(2+)-induced Ca2+ release channels (CICR) of sarcoplasmic reticulum (SR) in the cardiomyopathic hamster heart using [3H]PN-200 and [3H]ryanodine, respectively. Partially purified cardiac membrane preparations from myopathic animals exhibit a twofold higher capacity to bind both [3H]PN-200 and [3H]ryanodine. Crude particulate membrane fractions from normal and cardiomyopathic animals reveal no significant difference in receptor densities for [3H]PN-200, whereas densities for [3H]ryanodine binding sites and mRNA levels are significantly (P < 0.05) diminished in cardiomyopathic animals. Inhibition of [3H]ryanodine binding by either Ca2+ or Mg2+ (in mM) as well as temperature dependence for receptor activation for [3H]ryanodine (Q10) is not significantly different, whereas membranes isolated from cardiomyopathic hearts are 1.4-fold and threefold more sensitive to activation by doxorubicin and Ca2+ (in microM), respectively. Vesicles isolated from myopathic hearts are more sensitive to inhibition of Ca2+ uptake by doxorubicin. The higher densities of binding sites for [3H]PN-200 and [3H]ryanodine observed in partially purified membrane fractions from cardiomyopathic hearts are more likely the result of altered patterns with which T-tubule and CICR channels fractionate in preparations from cardiomyopathic hamster heart rather than transcriptional upregulation and may be a consequence of the deficiency in a dystrophin-associated glycoprotein recently identified. Downregulation and functional changes in CICR channels may alter SR Ca2+ transport and contribute to the progression of cardiomyopathy in the hamster.

Animals↗
PubMed1989

Differential effect of global ischemia on the ryanodine-sensitive and ryanodine-insensitive calcium uptake of cardiac sarcoplasmic reticulum.

The effect of ischemia on the function of cardiac sarcoplasmic reticulum (SR) was assessed by the calcium uptake rate of rat whole-heart homogenates in the presence of 10 mM oxalate. Previous studies have shown that this uptake is restricted to the SR. The contribution of the ryanodine-sensitive fractions of the SR to the total homogenate uptake was assessed by using 20 microM ruthenium red and 625 microM ryanodine to close the SR calcium release channel under previously established optimal conditions. Global ischemia of 10, 15, 30, and 60 minutes depressed homogenate calcium uptake rate 19 +/- 2%, 50 +/- 6%, 65 +/- 3%, and 81 +/- 5%, respectively. This decrease was not observed when the uptake rates were measured after closure of the calcium channel with ryanodine or ruthenium red. Similar results were obtained with a Langendorff in vitro perfusion preparation, in which calcium uptake was decreased 35 +/- 5%, 37 +/- 8%, 58 +/- 7%, and 64 +/- 4% after 10, 15, 30, and 60 minutes of ischemia, but no significant decrease was observed when homogenate uptake rates were measured in the presence of ryanodine. Thus, ischemia caused a depression in the calcium uptake rate of cardiac SR only when this activity was measured in the absence of SR calcium channel blockers. Reperfusion of ischemic hearts in a Langendorff preparation resulted in recovery of homogenate calcium uptake activity that correlated well with the return to sinus rhythm of the reperfused hearts. These reperfused hearts showed no change in the calcium uptake rate measured in the presence of ryanodine. These results suggest that the decrease in homogenate calcium uptake caused by ischemia is not due to a defect in calcium pumping capabilities but is due to an increased efflux through the ryanodine-sensitive calcium release channel of cardiac SR.

Alkaloids↗
PubMed1996

Divergent effects of ruthenium red and ryanodine on Ca2+/calmodulin-dependent phosphorylation of the Ca2+ release channel (ryanodine receptor) in cardiac sarcoplasmic reticulum.

In cardiac muscle, a Ca2+/calmodulin-dependent protein kinase (CaM kinase) associated with the sarcoplasmic reticulum (SR) is known to phosphorylate the membrane proteins phospholamban, Ca(2+)-ATPase, and Ca(2+)-release channel (ryanodine receptor). Phosphorylation of phospholamban and Ca(2+)-ATPase is recognized to stimulate Ca2+ sequestration by the SR but the functional consequence of Ca2+ channel phosphorylation has not been clearly established. In this study, we investigated the effects of the SR Ca(2+)-release inhibitor, ruthenium red (RR), and the SR Ca(2+)-release activator, ryanodine (at submicromolar concentrations), on CaM kinase-mediated phosphorylation of the Ca(2+)-cycling proteins in rabbit cardiac SR. Incubation of SR with RR (5-30 microM) for 3 min at 37 degrees C resulted in marked (up to 85%) inhibition of Ca2+ channel phosphorylation (50% inhibition with 15 +/- 2 microM RR) by the endogenous membrane-associated CaM kinase. Phosphorylation of the Ca2+ channel by exogenously added multifunctional alpha CaM kinase II was also inhibited similarly by RR. Phosphorylation of the Ca(2+)-ATPase by endogenous and exogenous CaM kinase was inhibited only modestly (25-30%) by RR, and phospholamban phosphorylation was unaffected by RR. The magnitude of RR-induced inhibition of Ca2+ channel phosphorylation did not differ appreciably at saturating or subsaturating concentrations of Ca2+ or calmodulin, and in the absence or presence of protein phosphatase inhibitors. In contrast to the effects of RR, low concentrations of ryanodine (0.25-1 microM) caused significant stimulation (up to approximately 50%) of Ca2+ channel phosphorylation but had no effect on Ca(2+)-ATPase and phospholamban phosphorylation. These findings suggest that interaction of RR with the ryanodine receptor induces a "nonphosphorylatable state" of the Ca(2+)-release channel, likely through a conformational change involving occlusion of the CaM kinase phosphorylation site. On the other hand, ryanodine binding to the receptor may serve to maintain an open, "phosphorylatable state" of the channel.

Animals↗
PubMed1992

Volatile anesthetics selectively alter [3H]ryanodine binding to skeletal and cardiac ryanodine receptors.

The effect of clinical concentrations of volatile anesthetics on ryanodine receptors of cardiac and skeletal muscle sarcoplasmic reticulum was evaluated using [3H]ryanodine binding. At 2 volume percent, halothane and enflurane stimulated binding to cardiac SR by 238% and 204%, respectively, while isoflurane had no effect. In contrast, halothane and enflurane had no effect on [3H]ryanodine binding to skeletal ryanodine receptors, while isoflurane produced a significant stimulation. These results suggest that volatile anesthetics interact in a site-specific manner with ryanodine receptors of cardiac or skeletal muscle to effect Ca2+ release-channel gating.

Animals↗
PubMed1999

Role of the ryanodine receptor in ischemic brain damage--localized reduction of ryanodine receptor binding during ischemia in hippocampus CA1.

1. The ryanodine receptor has recently been shown to play a pivotal role in the regulation of intracellular Ca2+ concentration via Ca(2+)-induced Ca2+ release (CICR). Effects of ischemia on CICR in the brain tissue, however, remain largely unknown since only a few reports have been published on this subject. In this paper we report on work in this area by our group and review related progress in this field. 2. We examined alterations of ryanodine receptor binding and local cerebral blood flow (LCBF) at 15 min, 30 min, and 2 hr after occlusion of the right common carotid artery in the gerbil brain. A quantitative autoradiographic method permitted simultaneous measurement of these parameters in the same brain. The LCBF was significantly reduced in most of the cerebral regions on the occluded side during each time period of ischemia. In contrast, only in the hippocampus CA1 on the occluded side was a significant reduction in ryanodine binding found at 15 min, 30 min and 2 hr after the occlusion. 3. These findings suggest that suppression of ryanodine binding in the hippocampus CA1 may be attributable to a regionally specific perturbation of CICR and that this perturbation may be closely associated with the pathophysiological mechanism that leads to be selective ischemic vulnerability of this region. 4. Other recent studies have also reported an important role for ryanodine receptors in neuronal injury such as the delayed neuronal death in the hippocampus CA1. These data suggest that derangement of CICR is likely to be involved in acute neuronal necrosis as well as in delayed neuronal death in ischemia. 5. Further studies on clarifying the role of CICR in ischemic brain damage are needed in order to develop new therapeutic strategies for stroke patients.

Animals↗
PubMed1992

Reversibility of the effects of normothermic global ischemia on the ryanodine-sensitive and ryanodine-insensitive calcium uptake of cardiac sarcoplasmic reticulum.

The effect of normothermic ischemia and ischemia/reperfusion on the function of cardiac sarcoplasmic reticulum (CSR) was investigated using a modified Langendorff perfusion of isolated rat hearts. The function of the CSR was assessed by the oxalate-supported Ca2+ uptake rate of ventricular homogenates. The contribution of the ryanodine-sensitive portion of the CSR was determined by using 20 microM ruthenium red or 625 microM ryanodine to close the CSR Ca2+ release channel. The Ca2+ uptake rate of the CSR decreased progressively with increasing duration of ischemia, but this depression was much less when uptake was assayed in the presence of ryanodine. The depression in CSR Ca2+ uptake preceded ischemic contracture. Ryanodine and ruthenium red stimulated uptake almost equally in control hearts, but ruthenium red was much less effective than ryanodine after ischemia. This difference could not be overcome by increasing the ruthenium red concentration. These results confirm the suggestion that the Ca2+ release channel is inappropriately opened after ischemia. The CSR uptake rates were almost completely restored at 15 minutes of reperfusion after 5 and 10 minutes of ischemia but were only partially restored after 15 minutes of ischemia. At reperfusion, mechanical function (end-diastolic pressure and peak systolic developed pressure) was markedly depressed after only 15 minutes of ischemia. The degree of "stunning" correlated well with the depression of CSR function in individual hearts. The decreased Ca2+ uptake of the CSR was not due to a buildup of ADP in the homogenates.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2006

Effects of an alpha-helical ryanodine receptor C-terminal tail peptide on ryanodine receptor activity: modulation by Homer.

We have determined the structure of a domain peptide corresponding to the extreme 19 C-terminal residues of the ryanodine receptor Ca2+ release channel. We examined functional interactions between the peptide and the channel, in the absence and in the presence of the regulatory protein Homer. The peptide was partly alpha-helical and structurally homologous to the C-terminal end of the T1 domain of voltage-gated K+ channels. The peptide (0.1-10 microM) inhibited skeletal ryanodine receptor channels when the cytoplasmic Ca2+ concentration was 1 microM; but with 10 microM cytoplasmic Ca2+, skeletal ryanodine receptors were activated by < or = 1.0 microM peptide and inhibited by 10 microM peptide. Cardiac ryanodine receptors on the other hand were inhibited by all peptide concentrations, at both Ca2+ concentrations. When channels did open in the presence of the peptide, they were more likely to open to substate levels. The inhibition and increased fraction of openings to subconductance levels suggested that the domain peptide might destabilise inter-domain interactions that involve the C-terminal tail. We found that Homer 1b not only interacts with the channels, but reduces the inhibitory action of the C-terminal tail peptide, perhaps by stabilizing inter-domain interactions and preventing their disruption.

Animals↗
PubMed1995

Peptide probe of ryanodine receptor function. Imperatoxin A, a peptide from the venom of the scorpion Pandinus imperator, selectively activates skeletal-type ryanodine receptor isoforms.

We have used [3H]ryanodine binding experiments and single channel recordings to provide convergent descriptions of the effect of imperatoxin A (IpTxa), a approximately 5-kDa peptide from the venom of the scorpion Pandinus imperator (Valdivia, H. H., Kirby, M. S., Lederer, W. J., and Coronado, R. (1992) Proc. Ntl. Acad. Sc. U.S.A. 89, 12185-12189) on Ca2+ release channels/ryanodine receptors (RyR) of sarcoplasmic reticulum (SR). At nanomolar concentrations, IpTxa increased the binding of [3H]ryanodine to skeletal SR and, to a lesser extent, to cerebellum microsomes. The activating effect of IpTxa on skeletal SR was Ca(2+)-dependent, synergized by caffeine, and independent of other modulators of RyRs. However, IpTxa had negligible effects on tissues where the expression of skeletal-type RyR isoforms (RyR1) is small or altogether absent, i.e. cardiac, cerebrum, and liver microsomes. Thus, IpTxa may be used as a ligand capable of discriminating between RyR isoforms with nanomolar affinity. IpTxa increased the open probability (Po) of rabbit skeletal muscle RyRs by increasing the frequency of open events and decreasing the duration of the closed lifetimes. This activating effect was dose-dependent (ED50 = 10 nM), had a fast onset, and was fully reversible. Purified RyR from solubilized skeletal SR displayed high affinity for [3H]ryanodine with a KD of 6.1 nM and Bmax of approximately 30 pmol/mg of protein. IpTxa increased [3H]ryanodine binding noncompetitively by increasing Bmax to approximately 60 pmol/mg of protein. These results suggested the presence of an IpTxa-binding site on the RyR or a closely associated regulatory protein. This site appears to be distinct from the caffeine- and adenine nucleotide-regulatory sites. IpTxa may prove a useful tool to identify regulatory domains critical for channel gating and to dissect the contribution of skeletal-type RyRs to intracellular Ca2+ waveforms generated by stimulation of different RyR isoforms.

Adenine Nucleotides↗
PubMed2000

Modulation of the calmodulin-induced inhibition of sarcoplasmic reticulum calcium release channel (ryanodine receptor) by sulfhydryl oxidation in single channel current recordings and [(3)H]ryanodine binding.

The modulation of the calmodulin-induced inhibition of the calcium release channel (ryanodine receptor) by two sulfhydryl oxidizing compounds, 4-(chloromercuri)phenyl-sulfonic acid (4-CMPS) and 4, 4'-dithiodipyridine (4,4'-DTDP) was determined by single channel current recordings with the purified and reconstituted calcium release channel from rabbit skeletal muscle sarcoplasmic reticulum (HSR) and [(3)H]ryanodine binding to HSR vesicles. 0.1 microm CaM reduced the open probability (P(o)) of the calcium release channel at maximally activating calcium concentrations (50-100 microm) from 0.502 +/- 0.02 to 0.137 +/- 0.022 (n = 28), with no effect on unitary conductance. 4-CMPS (10-40 microm) and 4,4'-DTDP (0.1-0.3 mm) induced a concentration dependent increase in P(o) (> 0.9) and caused the appearance of longer open states. CaM shifted the activation of the calcium release channel by 4-CMPS or 4,4'-DTDP to higher concentrations in single channel recordings and [(3)H]ryanodine binding. 40 microm 4-CMPS induced a near maximal (P(o) > 0.9) and 0.3 mm 4,4'-DTDP a submaximal (P(o) = 0.74) channel opening in the presence of CaM, which was reversed by the specific sulfhydryl reducing agent DTT. Neither 4-CMPS nor 4,4'-DTDP affected Ca-[(125)I]calmodulin binding to HSR. 1 mm MgCl(2) reduced P(o) from 0.53 to 0.075 and 20-40 microm 4-CMPS induced a near maximal channel activation (P(o) > 0.9). These results demonstrate that the inhibitory effect of CaM or magnesium in a physiological concentration is diminished or abolished at high concentrations of 4-CMPS or 4,4'-DTDP through oxidation of activating sulfhydryls on cysteine residues of the calcium release channel.

Animals↗
PubMed1996

Similar Ca2+ dependences of [3H]ryanodine binding to alpha- and beta-ryanodine receptors purified from bullfrog skeletal muscle in an isotonic medium.

To understand the functions of the two ryanodine receptor isoforms (alpha- and beta-RyRs) in nonmammalian skeletal muscles, we determined [3H]ryanodine binding to these isoforms purified from bullfrog skeletal muscle. In 0.17 M-NaCl medium both isoforms demonstrated similar Ca2+ dependent ryanodine binding activities, while the Ca2+ sensitivity for activation of beta-RyR was increased in 1 M-NaCl medium. This enhancement in Ca2+ sensitivity depended on the kinds of salts used. These results imply that alpha- and beta-RyRs may have similar properties as Ca2+-induced Ca2+ release channels in bullfrog skeletal muscle.

Adenosine Triphosphate↗
PubMed2001

Short- and long-term functional alterations of the skeletal muscle calcium release channel (Ryanodine receptor) by Suramin: apparent dissociation of single channel current recording and [3H]ryanodine binding.

The present study demonstrates the following characteristic suramin actions on the purified skeletal muscle calcium release channel in single-channel current recordings and [(3)H]ryanodine binding to HSR: 1) Suramin (0.3-0.9 mM) induced a concentration-dependent increase in the open probability (P(o) congruent with 0.9) at 20 to 100 microM Ca(2+) and an almost fully open channel at 1 mM Ca(2+) (P(o) = 0.95) with a marked shift to longer open states (tau(o)3/tau(o)4). Suramin increased the apparent calcium affinity to the activating high-affinity calcium binding sites and reduced the apparent magnesium affinity to the inhibitory low affinity Ca(2+)/Mg(2+) binding sites. 2) Channel activation by suramin and sulfhydryl oxidation was additive. 3) Suramin (0.9 mM) reversed the Ca-calmodulin-induced channel inhibition at 0.1 or 1 to 5 microM Ca-calmodulin. 4) The open probability of the suramin activated channel was almost completely inhibited by 10 mM Mg(2+) or Ca(2+) on short suramin exposure. Prolonged suramin exposure (30-60 min) resulted in a time-dependent, slow increase in P(o), with long open states of low frequency in the presence of 10 to 20 mM Mg(2+) or Ca(2+). 5) Magnesium induced inhibition of P(o) (IC(50) = 0.38 mM) and equilibrium [(3)H]ryanodine binding (IC(50) = 0.30 mM) agreed well in control channels, but were dissociated in the presence of 0.9 to 1.0 mM suramin (IC(50) = 0.82 mM versus 83 mM). [(3)H]ryanodine binding seemed to monitor predominantly the long-term alteration in channel function. 6) The multiple effects of suramin on channel function suggest an allosteric mechanism and no direct effects on binding of endogenous ligands involved in channel gating.

Animals↗
PubMed2001

A ryanodine fluorescent derivative reveals the presence of high-affinity ryanodine binding sites in the Golgi complex of rat sympathetic neurons, with possible functional roles in intracellular Ca(2+) signaling.

The plant alkaloid ryanodine (Ry) is a high-affinity modulator of ryanodine receptor (RyR) Ca(2+) release channels. Although these channels are present in a variety of cell types, their functional role in nerve cells is still puzzling. Here, a monosubstituted fluorescent Ry analogue, B-FL-X Ry, was used to reveal the distribution of RyRs in cultured rat sympathetic neurons. B-FL-X Ry competitively inhibited the binding of [3H]Ry to rabbit skeletal muscle SR membranes, with an IC(50) of 150 nM, compared to 7 nM of unlabeled Ry. Binding of B-FL-X Ry to the cytoplasm of sympathetic neurons is saturable, reversible and of high affinity. The pharmacology of B-FL-X Ry showed marked differences with unlabeled Ry, which are partially explained by its lower affinity: (1) use-dependent reversible inhibition of caffeine-induced intracellular Ca(2+) release; (2) diminished voltage-gated Ca(2+) influx, due to a positive shift in the activation of voltage gated Ca(2+) currents. B-FL-X Ry-stained sympathetic neurons, viewed under confocal microscopy, showed conspicuous labeling of crescent-shaped structures pertaining to the Golgi complex, a conclusion supported by experiments showing co-localization with Golgi-specific fluorescent probes and the breaking up of crescent-shaped staining after treatment with drugs that disassemble Golgi complex. The presence of RyRs to the Golgi could be confirmed with specific anti-RyR(2) antibodies, but evidence of caffeine-induced Ca(2+) release from this organelle could not be obtained using fast confocal microscopy. Rather, an apparent decrease of the cytosolic Ca(2+) signal was detected close to this organelle. In spite of that, short-term incubation with brefeldin A (BFA) suppressed the fast component of caffeine-induced Ca(2+) release, and the Ca(2+) release process lasted longer and appeared less organized. These observations, which suggest a possible role of the Golgi complex in Ca(2+) homeostasis and signaling in nerve cells, could be relevant to reports involving derangement of the Golgi complex as a probable cause of some forms of progressive neuronal degeneration, such as Alzheimer's disease and amyotrophic lateral sclerosis.

Animals↗
PubMed2000

Depletion of intracellular Ca2+ by caffeine and ryanodine induces apoptosis of chinese hamster ovary cells transfected with ryanodine receptor.

Recent studies have suggested a central role for Ca(2+) in the signaling pathway of apoptosis and certain anti-apoptotic effects of Bcl-2 family of proteins have been attributed to changes in intracellular Ca(2+) homeostasis. Here we report that depletion of Ca(2+) from endoplasmic reticulum (ER) leads to apoptosis in Chinese hamster ovary cells. Stable expression of ryanodine receptor (RyR) in these cells enables rapid and reversible changes of both cytosolic Ca(2+) and ER Ca(2+) content via activation of the RyR/Ca(2+) release channel by caffeine and ryanodine. Sustained depletion of the ER Ca(2+) store leads to apoptosis in Chinese hamster ovary cells, whereas co-expression of Bcl-xL and RyR in these cells prevents apoptotic cell death but not necrotic cell death. The anti-apoptotic effect of Bcl-xL does not correlate with changes in either the Ca(2+) release process from the ER or the capacitative Ca(2+) entry through the plasma membrane. The data suggest that Bcl-xL likely prevents apoptosis of cells at a stage downstream of ER Ca(2+) release and capacitative Ca(2+) entry.

Animals↗
PubMed1999

Malignant hyperthermia causing Gly2435Arg mutation of the ryanodine receptor facilitates ryanodine-induced calcium release in myotubes.

We have investigated if cultivated muscle cells from malignant hyperthermia (MH) patients can be distinguished pharmacologically from controls. Muscle specimens from four individuals carrying the Gly2435Arg mutation of the skeletal muscle ryanodine receptor protein (RYR1) and from four controls were used to culture myotubes. Resting intracellular calcium concentration ([Ca2+]i) of MH myotubes was similar to controls. However, when ryanodine 0.5 mumol litre-1 was added, the kinetics of the increase in the calcium signals in MH and control cells were significantly different; the time for half maximum increase was mean 197 (SD 131) s for MH cells and 474 (61) s for controls (n = 80 cells each). On average, the area under the MH response curves was twice the control value. These results give rise to hopes that the phenotype of MH can be characterized using cultured human muscle and that a culture-based test for MH susceptibility may eventually be developed.

Adolescent↗
PubMed2005

Multiple ryanodine receptor subtypes and heterogeneous ryanodine receptor-gated Ca2+ stores in pulmonary arterial smooth muscle cells.

Ryanodine receptors (RyRs) of pulmonary arterial smooth muscle cells (PASMCs) play important roles in major physiological processes such as hypoxic pulmonary vasoconstriction and perinatal pulmonary vasodilatation. Recent studies show that three subtypes of RyRs are coexpressed and RyR-gated Ca2+ stores are distributed heterogeneously in systemic vascular myocytes. However, the molecular identity and subcellular distribution of RyRs have not been examined in PASMCs. In this study we detected mRNA and proteins of all three subtypes in rat intralobar PASMCs using RT-PCR and Western blot. Quantitative real-time RT-PCR showed that RyR2 mRNA was most abundant, approximately 15-20 times more than the other two subtypes. Confocal fluorescence microscopy revealed that RyRs labeled with BODIPY TR-X ryanodine were localized in the peripheral and perinuclear regions and were colocalized with sarcoplasmic reticulum labeled with Fluo-5N. Immunostaining showed that the subsarcolemmal regions exhibited clear signals of RyR1 and RyR2, whereas the perinuclear compartments contained mainly RyR1 and RyR3. Ca2+ sparks were recorded in both regions, and their activities were enhanced by a subthreshold concentration of caffeine or by endothelin-1, indicating functional RyR-gated Ca2+ stores. Moreover, 18% of the perinuclear sparks were prolonged [full duration/half-maximum (FDHM) = 193.3 +/- 22.6 ms] with noninactivating kinetics, in sharp contrast to the typical fast inactivating Ca2+ sparks (FDHM = 44.6 +/- 3.2 ms) recorded in the same PASMCs. In conclusion, multiple RyR subtypes are expressed differentially in peripheral and perinuclear RyR-gated Ca2+ stores; the molecular complexity and spatial heterogeneity of RyRs may facilitate specific Ca2+ regulation of cellular functions in PASMCs.

Animals↗