Kinetics of neurotransmitter release in neuromuscular synapses of newborn and adult rats.

The kinetics of the phasic synchronous and delayed asynchronous release of acetylcholine quanta was studied at the neuromuscular junctions of aging rats from infant to mature animals at various frequencies of rhythmic stimulation of the motor nerve. We found that in infants 6 (P6) and 10 (P10) days after birth a strongly asynchronous phase of quantal release was observed, along with a reduced number of quanta compared to the synapses of adults. The rise time and decay of uni-quantal end-plate currents were significantly longer in infant synapses. The presynaptic immunostaining revealed that the area of the synapses in infants was significantly (up to six times) smaller than in mature junctions. The intensity of delayed asynchronous release in infants increased with the frequency of stimulation more than in adults. A blockade of the ryanodine receptors, which can contribute to the formation of delayed asynchronous release, had no effect on the kinetics of delayed secretion in the infants unlike synapses of adults. Therefore, high degree of asynchrony of quantal release in infants is not associated with the activity of ryanodine receptors and with the liberation of calcium ions from intracellular calcium stores.

Age-related regulation of excitation–contraction coupling in rat heart

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Hearts from subjects with different ages have different Ca2+ signaling. Release of Ca2+ from intracellular stores in response to an action potential initiates cardiac contraction. Both depolarization-stimulated and spontaneous Ca2+ releases, Ca2+ transients and Ca2+ sparks, demonstrate the main events of excitation–contraction coupling (ECC). Global increase in free Ca2+concentration ([Ca2+] i ) consists of summation of Ca2+ release events in cardiomyocytes. Since the Ca2+ flux induced by Ca2+ sparks reports a summation of ryanodine-sensitive Ca2+ release channels (RyR2s)’s behavior in a spark cluster, evaluation of the properties of Ca2+ sparks and Ca2+ transients may provide insight into the role of RyR2s on altered heart function between 3-month-old (young adult) and 6-month-old (mature adult) rats. Basal [Ca2+] i and Ca2+ sparks frequency were significantly higher in mature adult rats compared to those of young adults. Moreover, amplitudes of Ca2+ sparks and Ca2+ transients were significantly smaller in mature adults than those of young adults with longer time courses. A smaller L-type Ca2+ current density and decreased SR Ca2+ load was observed in mature adult rats. In addition, RyR2s were markedly hyperphosphorylated, and phosphorylation levels of PKA and CaMKII were higher in heart from mature adults compared to those of young adults, whereas their SERCA protein levels were similar. Our data demonstrate that hearts from rats with different ages have different Ca2+ signaling including hyperphosphorylation of RyR2s and higher basal [Ca2+] i together with increased oxidized protein-thiols in mature adult rats compared to those of young adults, which play important roles in ECC. Finally, we report that ECC efficiency changes with age during maturation, partially related with an increased cellular oxidation level leading to reduced free protein-thiols in cardiomyocytes (AU)

The mitochondrial ryanodine receptor in rat heart: a pharmaco-kinetic profile.

A protein discovered within inner mitochondrial membranes (IMM), designated as the mitochondrial ryanodine receptor (mRyR), has been recognized recently as a modulator of Ca(2+) fluxes in mitochondria. The present study provides fundamental pharmacological and electrophysiological properties of this mRyR. Rat cardiac IMM fused to lipid bilayers revealed the presence of a mitochondrial channel with gating characteristics similar to those of classical sarcoplasmic reticulum RyR (SR-RyR), but a variety of other mitochondrial channels obstructed clean recordings. Mitochondrial vesicles were thus solubilized and subjected to sucrose sedimentation to obtain mRyR-enriched fractions. Reconstitution of sucrose-purified fractions into lipid bilayers yielded Cs(+)-conducting, Ca(2+)-sensitive, large conductance (500-800 pS) channels with signature properties of SR-RyRs. Cytosolic Ca(2+) increased the bursting frequency and mean open time of the channel. Micromolar concentrations of ryanodine induced the appearance of subconductance states or inhibited channel activity altogether, while Imperatoxin A (IpTx(a)), a specific activator of RyRs, reversibly induced the appearance of distinct subconductance states. Remarkably, the cardiac mRyR displayed a Ca(2+) dependence of [(3)H]ryanodine binding curve similar to skeletal RyR (RyR1), not cardiac RyR (RyR2). Overall, the mRyR displayed elemental attributes that are present in single channel lipid bilayer recordings of SR-RyRs, although some exquisite differences were also noted. These results therefore provide the first direct evidence that a unique RyR occurs in mitochondrial membranes.

Ryanodine receptor type 1 (RyR1) possessing malignant hyperthermia mutation R615C exhibits heightened sensitivity to dysregulation by non-coplanar 2,2',3,5',6-pentachlorobiphenyl (PCB 95).

Malignant hyperthermia (MH) susceptibility is conferred by inheriting one of >60 missense mutations within the highly regulated microsomal Ca(2+) channel known as ryanodine receptor type 1 (RyR1). Although MH susceptible patients lack overt clinical signs, a potentially lethal MH syndrome can be triggered by exposure to halogenated alkane anesthetics. This study compares how non-coplanar 2,2',3,5',6-pentachlorobiphenyl (PCB 95), a congener identified in environmental and human samples, alters the binding properties of [(3)H]ryanodine to RyR1 in vitro. Junctional sarcoplasmic reticulum (SR) was isolated from skeletal muscle dissected from wild type pigs ((Wt)RyR1) and pigs homozygous for MH mutation R615C ((MH)RyR1), a mutation also found in humans. Although the level of (Wt)RyR1 and (MH)RyR1 expression is the same, (MH)RyR1 shows heightened sensitivity to activation and altered regulation by physiological cations. We report here that (MH)RyR1 shows more pronounced activation by Ca(2+), and is less sensitive to channel inhibition by Ca(2+) and Mg(2+), compared to (Wt)RyR1. In a buffer containing 100nM free Ca(2+), conditions typically found in resting cells, PCB 95 (50-1000nM) enhances the activity of (MH)RyR1 whereas it has no detectable effect on (Wt)RyR1. PCB 95 (2microM) decreases channel inhibition by Mg(2+) to a greater extent in (MH)RyR1 (IC(50) increased nine-fold) compared to (Wt)RyR1 (IC(50) increased by 2.5-fold). PCB95 reduces inhibition by Ca(2+) two-fold more with (MH)RyR1 than (Wt)RyR1. Our data suggest that non-coplanar PCBs are more potent and efficacious toward (MH)RyR1 than (Wt)RyR1, and have more profound effects on its cation regulation. Considering the important roles of Ca(2+) and Mg(2+) in regulating Ca(2+) signals involving RyR channels, these data provide the first mechanistic evidence that a genetic mutation known to confer susceptibility to pharmacological agents also enhances sensitivity to an environmental contaminant.

Effects of low-intensity training on dihydropyridine and ryanodine receptor content in skeletal muscle of mouse

To evaluate low-intensity exercise training induced changes in the expression of dihydropyridine (DHP) and ryanodine (Ry) receptors both mRNA and protein levels were determined by quantitative RT-PCR and immunoblot analysis from gastrocnemius (GAS) and rectus femoris (RF) muscles of mice subjected to a 15-week aerobic exercise program. The level of muscular work was assayed by changes in myosin heavy chain (MHC) content, myoglobin (Mb) expression and muscle size. The mRNA expression and optical density of DHP receptor increased significantly in GAS by 66.8 and 39.5%, respectively. The expression of Ry receptor, on the other hand, was not up-regulated. In RF, there was a significant increase of 38.4% in the mRNA expression of DHP receptor, although the protein level remained the same. No changes in Ry receptor expression was observed. The training resulted in a 1.58% increase in the amount of MHC IIa and a 2.34% decrease in that of IIb and IId in GAS. A significant 8.3% increase in the Mb content was observed. In RF, no significant changes in MHC or in Mb content were noted. Our results show that an evident increase in the mRNA and protein expression of DHP receptor was induced in GAS even by a relatively low-intensity exercise. Surprisingly, contrast to DHP receptor expression, no changes in Ry receptor mRNA, or protein levels were found, indicating more abundant demand for DHP receptor after increased muscle activity (AU)

Para evaluar los cambios inducidos en la expresión de los receptores de dihidropiridina (DHPR) y rianodina (RyR) por el entrenamiento con ejercicio de baja intensidad, se determinan los niveles de mRNA y de proteína mediante el análisis de RT-PCR cuantitativa e inmunoblot de los músculos gastrocnemius (GAS) y rectus femoris (RF) de ratón sometido a un programa de ejercicio aeróbico durante 15 semanas. El nivel de trabajo muscular fue determinado por los cambios en contenido de cadena pesada de miosina (MHC), expresión de mioglobina (Mb) y tamaño del músculo. La cantidad de mRNA y de proteína de DHPR aumentó significativamente en un 66,8 y 39,5% respectivamente. La expresión de RyR, por otro lado, no se vio incrementada. En RF hubo un aumento significativo del 22,7% en la expresión del mRNA de DHPR, aunque los niveles de proteína permanecieron inalterados. Tampoco se observaron cambios en la expresión de RyR en RF. El entrenamiento dio lugar a un aumento del 1,58% en la cantidad de MHC IIa y disminución del 2,34% en MHC IIb y IId en GAS, con incremento significativo del 8,3% en el contenido de Mb. En RF no se detectaron cambios significativos en el contenido en MHC ni en Mb. Nuestros resultados muestran que se induce un evidente aumento en el nivel de RNAm y de proteína DHPR en GAS mediante un ejercicio de relativamente baja intensidad. Sorprendentemente, en contraste con la expresión de DHPR, no se encontraron cambios en los niveles de mRNA ni de proteína de RyR, indicando mayor demanda de DHPR al incrementar la actividad muscular (AU)

Flubendiamide, a novel Ca2+ channel modulator, reveals evidence for functional cooperation between Ca2+ pumps and Ca2+ release.

Flubendiamide, developed by Nihon Nohyaku Co., Ltd. (Tokyo, Japan), is a novel activator of ryanodine-sensitive calcium release channels (ryanodine receptors; RyRs), and is known to stabilize insect RyRs in an open state in a species-specific manner and to desensitize the calcium dependence of channel activity. In this study, using flubendiamide as an experimental tool, we examined an impact of functional modulation of RyR on Ca2+ pump. Strikingly, flubendiamide induced a 4-fold stimulation of the Ca2+ pump activity (EC50=11 nM) of an insect that resequesters Ca2+ to intracellular stores, a greater increase than with the classical RyR modulators ryanodine and caffeine. This prominent stimulation, which implies tight functional coupling of Ca2+ release with Ca2+ pump, resulted in a marginal net increase in the extravesicular calcium concentration despite robust Ca2+ release from the intracellular stores by flubendiamide. Further analysis suggested that luminal Ca2+ is an important mediator for the functional coordination of RyRs and Ca2+ pumps. However, kinetic factors for Ca2+ pumps, including ATP and cytoplasmic Ca2+, failed to affect the Ca2+ pump stimulation by flubendiamide. We therefore conclude that the stimulation of Ca2+ pump by flubendiamide is mediated by the decrease in luminal calcium, which may induce calcium dissociation from the luminal Ca2+ binding site on the Ca2+ pump. This mechanism should play an essential role in precise control of intracellular Ca2+ homeostasis.

Location of ryanodine and dihydropyridine receptors in frog myocardium.

Frog myocardium depends almost entirely on calcium entry from extracellular spaces for its beat-to-beat activation. Atrial myocardium additionally shows internal calcium release under certain conditions, but internal release in the ventricle is absent or very low. We have examined the content and distribution of the sarcoplasmic reticulum (SR) calcium release channels (ryanodine receptors, RyRs) and the surface membrane calcium channels (dihydropyridine receptors, DHPRs) in myocardium from the two atria and the ventricle of the frog heart using binding of radioactive ryanodine, immunolabeling of RyR and DHPR, and thin section and freeze-fracture electron microscopy. In cells from both types of chambers, the SR forms peripheral couplings and in both chambers peripheral couplings colocalize with clusters of DHPRs. However, although a low level of high affinity binding of ryanodine is detectable and RyRs are present in peripheral couplings of the atrium, the ventricle shows essentially no ryanodine binding and RyRs are not detectable either by electron microscopy or immunolabeling. The results are consistent with the lack of internal calcium release in the ventricle, and raise questions regarding the significance of DHPR at peripheral couplings in the absence of RyR. Interestingly, the free SR membrane in both heart chambers shows a low but equal density of intramembrane particles representing the Ca(2+) ATPase.

Identification of a ryanodine receptor in rat heart mitochondria.

Recent studies have shown that, in a wide variety of cells, mitochondria respond dynamically to physiological changes in cytosolic Ca(2+) concentrations ([Ca(2+)](c)). Mitochondrial Ca(2+) uptake occurs via a ruthenium red-sensitive calcium uniporter and a rapid mode of Ca(2+) uptake. Surprisingly, the molecular identity of these Ca(2+) transport proteins is still unknown. Using electron microscopy and Western blotting, we identified a ryanodine receptor in the inner mitochondrial membrane with a molecular mass of approximately 600 kDa in mitochondria isolated from the rat heart. [(3)H]Ryanodine binds to this mitochondrial ryanodine receptor with high affinity. This binding is modulated by Ca(2+) but not caffeine and is inhibited by Mg(2+) and ruthenium red in the assay medium. In the presence of ryanodine, Ca(2+) uptake into isolated heart mitochondria is suppressed. In addition, ryanodine inhibited mitochondrial swelling induced by Ca(2+) overload. This swelling effect was not observed when Ca(2+) was applied to the cytosolic fraction containing sarcoplasmic reticulum. These results are the first to identify a mitochondrial Ca(2+) transport protein that has characteristics similar to the ryanodine receptor. This mitochondrial ryanodine receptor is likely to play an essential role in the dynamic uptake of Ca(2+) into mitochondria during Ca(2+) oscillations.

Classes of thiols that influence the activity of the skeletal muscle calcium release channel.

The skeletal muscle Ca(2+) release channel/ryanodine receptor (RyR1) is a prototypic redox-responsive ion channel. Nearly half of the 101 cysteines per RyR1 subunit are kept in a reduced (free thiol) state under conditions comparable with resting muscle. Here we assessed the effects of physiological determinants of cellular redox state (oxygen tension, reduced (GSH) or oxidized (GSSG) glutathione, and NO/O(2) (released by 3-morpholinosydnonimine)) on RyR1 redox state and activity. Oxidation of approximately 10 RyR1 thiols (from approximately 48 to approximately 38 thiols/RyR1 subunit) had little effect on channel activity. Channel activity increased reversibly as the number of thiols was further reduced to approximately 23/subunit, whereas more extensive oxidation (to approximately 13 thiols/subunit) inactivated the channel irreversibly. Neither S-nitrosylation nor tyrosine nitration contributed to these effects. The results identify at least three functional classes of RyR1 thiols and suggest that 1) the channel may be protected from oxidation by a large reservoir of functionally inert thiols, 2) the channel may be designed to respond to moderate oxidative stress by a change in activation setpoint, and 3) the channel is susceptible to oxidative injury under more extensive conditions.

Evidence for a role of the lumenal M3-M4 loop in skeletal muscle Ca(2+) release channel (ryanodine receptor) activity and conductance.

We tested the hypothesis that part of the lumenal amino acid segment between the two most C-terminal membrane segments of the skeletal muscle ryanodine receptor (RyR1) is important for channel activity and conductance. Eleven mutants were generated and expressed in HEK293 cells focusing on amino acid residue I4897 homologous to the selectivity filter of K(+) channels and six other residues in the M3-M4 lumenal loop. Mutations of amino acids not absolutely conserved in RyRs and IP(3)Rs (D4903A and D4907A) showed cellular Ca(2+) release in response to caffeine, Ca(2+)-dependent [(3)H]ryanodine binding, and single-channel K(+) and Ca(2+) conductances not significantly different from wild-type RyR1. Mutants with an I4897 to A, L, or V or D4917 to A substitution showed a decreased single-channel conductance, loss of high-affinity [(3)H]ryanodine binding and regulation by Ca(2+), and an altered caffeine-induced Ca(2+) release in intact cells. Mutant channels with amino acid residue substitutions that are identical in the RyR and IP(3)R families (D4899A, D4899R, and R4913E) exhibited a decreased K(+) conductance and showed a loss of high-affinity [(3)H]ryanodine binding and loss of single-channel pharmacology but maintained their response to caffeine in a cellular assay. Two mutations (G4894A and D4899N) were able to maintain pharmacological regulation both in intact cells and in vitro but had lower single-channel K(+) and Ca(2+) conductances than the wild-type channel. The results support the hypothesis that amino acid residues in the lumenal loop region between the two most C-terminal membrane segments constitute a part of the ion-conducting pore of RyR1.

Enhanced sarcoplasmic reticulum Ca(2+) release following intermittent sprint training.

To evaluate the effect of intermittent sprint training on sarcoplasmic reticulum (SR) function, nine young men performed a 5 wk high-intensity intermittent bicycle training, and six served as controls. SR function was evaluated from resting vastus lateralis muscle biopsies, before and after the training period. Intermittent sprint performance (ten 8-s all-out periods alternating with 32-s recovery) was enhanced 12% (P < 0.01) after training. The 5-wk sprint training induced a significantly higher (P < 0.05) peak rate of AgNO(3)-stimulated Ca(2+) release from 709 (range 560-877; before) to 774 (596-977) arbitrary units Ca(2+). g protein(-1). min(-1) (after). The relative SR density of functional ryanodine receptors (RyR) remained unchanged after training; there was, however, a 48% (P < 0.05) increase in total number of RyR. No significant differences in Ca(2+) uptake rate and Ca(2+)-ATPase capacity were observed following the training, despite that the relative density of Ca(2+)-ATPase isoforms SERCA1 and SERCA2 had increased 41% and 55%, respectively (P < 0.05). These data suggest that high-intensity training induces an enhanced peak SR Ca(2+) release, due to an enhanced total volume of SR, whereas SR Ca(2+) sequestration function is not altered.

Mechanisms of P(i) regulation of the skeletal muscle SR Ca(2+) release channel.

Inorganic phosphate (P(i)) accumulates in the fibers of actively working muscle where it acts at various sites to modulate contraction. To characterize the role of P(i) as a regulator of the sarcoplasmic reticulum (SR) calcium (Ca(2+)) release channel, we examined the action of P(i) on purified SR Ca(2+) release channels, isolated SR vesicles, and skinned skeletal muscle fibers. In single channel studies, addition of P(i) to the cis chamber increased single channel open probability (P(o); 0.079 +/- 0.020 in 0 P(i), 0. 157 +/- 0.034 in 20 mM P(i)) by decreasing mean channel closed time; mean channel open times were unaffected. In contrast, the ATP analog, beta,gamma-methyleneadenosine 5'-triphosphate (AMP-PCP), enhanced P(o) by increasing single channel open time and decreasing channel closed time. P(i) stimulation of [(3)H]ryanodine binding by SR vesicles was similar at all concentrations of AMP-PCP, suggesting P(i) and adenine nucleotides act via independent sites. In skinned muscle fibers, 40 mM P(i) enhanced Ca(2+)-induced Ca(2+) release, suggesting an in situ stimulation of the release channel by high concentrations of P(i). Our results support the hypothesis that P(i) may be an important endogenous modulator of the skeletal muscle SR Ca(2+) release channel under fatiguing conditions in vivo, acting via a mechanism distinct from adenine nucleotides.

Ca2+ release induced by myotoxin alpha, a radio-labellable probe having novel Ca2+ release properties in sarcoplasmic reticulum.

1. Myotoxin alpha (MYTX), a polypeptide toxin purified from the venom of prairie rattlesnakes (Crotalus viridis viridis) induced Ca2+ release from the heavy fraction (HSR) but not the light fraction of skeletal sarcoplasmic reticulum at concentrations higher than 1 microM, followed by spontaneous Ca2+ reuptake by measuring extravesicular Ca2+ concentrations using the Ca2+ electrode. 2. The rate of 45Ca2+ release from HSR vesicles was markedly accelerated by MYTX in a concentration-dependent manner in the range of concentrations between 30 nM and 10 microM, indicating the most potent Ca2+ releaser in HSR. 3. The Ca2+ dependency of MYTX-induced 45Ca2+ release has a bell-shaped profile but it was quite different from that of caffeine, an inducer of Ca(2+)-induced Ca2+ release. 4. 45Ca2+ release induced by MYTX was remarkable in the range of pCa between 8 and 3, whereas that by caffeine was prominent in the range of pCa, i.e., between 7 and 5.5. 5. MYTX-induced 45Ca2+ release consists of both early and late components. The early component caused by MYTX at low concentrations (30-300 nM) completed within 20 s, while the late component induced by it at higher concentrations (> 0.3 microM) was maintained for at least 1 min. 6. Both the components were almost completely inhibited by inhibitors of Ca2+ such as Mg2+, ruthenium red and spermine. 7. 45Ca2+ release induced by caffeine or beta,gamma-methyleneadenosine 5'-triphosphate (AMP-PCP) was completely inhibited by high concentrations of procaine. Procaine abolished the early component but not the late one, suggesting that at least the early component is mediated through Ca(2+)-induced Ca2+ release channels. 8. On the basis of these results, the character of Ca2+ release induced by MYTX was quite different from that caused by caffeine or AMP-PCP, suggesting that MYTX induces Ca2+ release having novel properties in HSR. MYTX is the first polypeptide Ca2+ inducer and has become a useful pharmacological tool for clarifying the mechanism of Ca2+ release from skeletal muscle SR.