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Japanese Journal of Pharmacology Dec 1990Properties of the intracellular Ca store were studied using saponin-skinned fiber bundles of guinea pig smooth muscles using a fluorescent Ca indicator method. There... (Review)
Review
Properties of the intracellular Ca store were studied using saponin-skinned fiber bundles of guinea pig smooth muscles using a fluorescent Ca indicator method. There exist two Ca release mechanisms in the Ca store: Ca-induced Ca release (CICR) and inositol 1,4,5-trisphosphate (IP3)-induced Ca release (IICR) mechanisms. The smooth muscle Ca store consists of two compartments: one (S alpha) has both CICR and IICR, and the other (S beta) has only IICR. The smooth muscle CICR is activated by greater than 1 microM Ca2+, has essentially the same properties with that in striated muscles, and is open-locked by ryanodine. After ryanodine treatment, therefore, the Ca uptake capacity of S alpha is selectively lost ('functional removal') with no effect on S beta. The IICR is Ca2(+)-dependent: Ca2+ enhances the IICR below 300 nM, but has also an inhibitory effect above this concentration. Therefore, Ca2+ acts on the IICR in a positive feedback manner when muscle tension is about to rise, making IP3 more effective, but this feedback is cut off as the tension approaches the maximum. ATP enhances the IICR as is the case in the CICR. 'Functional removal' of S alpha in intact bundles by ryanodine was used to estimate the role of Ca release in agonist-induced contractions. Ca release from the S alpha is important at least in the initial phase of contractions; and in the pulmonary artery, most of the activator Ca2+ originates from S alpha.
Topics: Animals; Calcium; Guinea Pigs; Inositol 1,4,5-Trisphosphate; Muscle Contraction; Muscle, Smooth; Nucleotides; Ryanodine; Saponins
PubMed: 2086998
DOI: 10.1254/jjp.54.345 -
British Journal of Pharmacology Aug 19901. The effects of ryanodine on changes in cytoplasmic Ca2+ level ([Ca2+]i) and muscle tension induced by maximum concentrations of phenylephrine (Phe; 1 microM),...
Ryanodine reveals multiple contractile and relaxant mechanisms in vascular smooth muscle: simultaneous measurements of mechanical activity and of cytoplasmic free Ca2+ level with fura-2.
1. The effects of ryanodine on changes in cytoplasmic Ca2+ level ([Ca2+]i) and muscle tension induced by maximum concentrations of phenylephrine (Phe; 1 microM), prostaglandin F2 alpha (PGF2 alpha, 10 microM), caffeine (Caf, 30 mM) and isoprenaline (Iso, 1 microM) were examined in rat aortic strips using fura-2. 2. In normal media, Phe and PGF2 alpha produced a phasic contraction, followed by a tonic one. Caf elicited only a transient contraction. When the preparation was treated with 10 microM ryanodine, an increase in [Ca2+]i was induced accompanied by a nicardipine (1 microM)-resistant contraction which was [Ca2+]o-dependent. 3. In Ca2(+)-free solution, the three stimulants elicited transient increases in [Ca2+]i. Transient contractions to Phe and Caf were accompanied by changes in [Ca2+]i. The transient increase in [Ca2+]i induced by PGF2 alpha was not accompanied by a corresponding contraction. 4. Sustained contractions were induced by Phe and PGF2 alpha in the absence of external Ca2+, while the increase in [Ca2+]i was reduced. A larger maximum contraction was induced by PGF alpha than by Phe. 5. Ryanodine abolished both the Caf- and Phe-induced [Ca2+]i transient increases and the corresponding contractions, but had no substantial effect on the PGF2 alpha-induced [Ca2+]i transient increase. Ryanodine had no influence on the sustained contractions induced by Phe and PGF2 alpha. 6. Iso relaxed both sustained contractions almost completely, without any detectable change in [Ca2+]i. Treatment of the preparation with ryanodine had no effect on the concentration-response curves for Iso in relaxing the 0.1 microM Phe- or 40 mM K(+)-induced precontraction. 7. It is suggested that Phe and Caf mobilize Ca2 + from a ryanodine-sensitive Ca2 + store and that PGF2 alpha. releases Ca2+ from a ryanodine-insensitive Ca2+ store. The former contributes to the transient contraction through a Ca2'-dependent process, while the latter seems not to be directly associated with the contraction. The sustained contraction under Ca2+-free conditions might involve a Ca2 '-independent process or a change in the sensitivity of the contractile filaments to Ca2 + 8. In addition to lowering cytoplasmic Ca2+ concentration, it is suggested that Iso counteracts the apparently Ca2 +-independent process. The ryanodine-sensitive Ca2+ store plays no substantial role in active relaxation by Iso, although it does play a major role in the maintenance of cytoplasmic Ca2+ in a quiescent muscle.
Topics: Animals; Caffeine; Calcium; Cytoplasm; Dinoprost; Egtazic Acid; Fura-2; Isoproterenol; Male; Muscle Contraction; Muscle Relaxation; Muscle, Smooth, Vascular; Phenylephrine; Rats; Rats, Inbred Strains; Ryanodine; Vasodilation
PubMed: 2119840
DOI: 10.1111/j.1476-5381.1990.tb14075.x -
Journal of Neurotrauma Feb 2022Ryanodine receptors (RyRs) mediate calcium release from calcium stores and have been implicated in axonal degeneration. Here, we use an intravital imaging approach to...
Ryanodine receptors (RyRs) mediate calcium release from calcium stores and have been implicated in axonal degeneration. Here, we use an intravital imaging approach to determine axonal fate after spinal cord injury (SCI) in real-time and assess the efficacy of ryanodine receptor inhibition as a potential therapeutic approach to prevent intra-axonal calcium-mediated axonal degeneration. Adult 6-8 week old YFP transgenic mice that express YFP in axons, as well as triple transgenic -Cre:Ai9:Ai95 mice that express the genetically-encoded calcium indicator GCaMP6f in tdTomato positive axons, were used to visualize axons and calcium changes in axons, respectively. Mice received a mild SCI at the T12 level of the spinal cord. Ryanodine, a RyR antagonist, was given at a concentration of 50 μM intrathecally within 15 min of SCI or delayed 3 h after injury and compared with vehicle-treated mice. RyR inhibition within 15 min of SCI significantly reduced axonal spheroid formation from 1 h to 24 h after SCI and increased axonal survival compared with vehicle controls. Delayed ryanodine treatment increased axonal survival and reduced intra-axonal calcium levels at 24 h after SCI but had no effect on axonal spheroid formation. Together, our results support a role for RyR in secondary axonal degeneration.
Topics: Animals; Axons; Calcium; Disease Models, Animal; Intravital Microscopy; Mice; Mice, Transgenic; Ryanodine; Ryanodine Receptor Calcium Release Channel; Spinal Cord; Spinal Cord Injuries
PubMed: 34913747
DOI: 10.1089/neu.2021.0350 -
Japanese Journal of Pharmacology Dec 1989We compared the effects of ryanodine and 9,21-didehydroryanodine (DH-ryanodine), which are present in commercial preparations of 'ryanodine', on the contractions of rat...
We compared the effects of ryanodine and 9,21-didehydroryanodine (DH-ryanodine), which are present in commercial preparations of 'ryanodine', on the contractions of rat and guinea pig aortae induced by 20 mM caffeine and tested the dependence of the action of each substance on external Ca2+. With the first protocol, the aortae were incubated with ryanodine or DH-ryanodine for 20 min in Ca2(+)-containing medium, and caffeine was added at 2 min incubation in Ca2(+)-free medium. With the second protocol, each substance was added when the external medium was changed to Ca2(+)-free medium, and 20 min later, caffeine was applied. Ryanodine and DH-ryanodine inhibited the caffeine-induced contractions in a similar way; i.e., with maximal effects at 3 microM and lesser effects at 10 microM. The potencies of inhibition by both substances were similar except that the effect of ryanodine at 1.5 microM was more potent than that of DH-ryanodine with the second protocol. The response by muscles previously loaded with Ca2+ to a second application of caffeine was more greatly inhibited by both compounds (use-dependent effect). The inhibition of the contraction due to the first or second application of caffeine was greater when either agent was applied in Ca2+-containing medium than in Ca2(+)-free medium. These results indicate that ryanodine and DH-ryanodine are similar in their effects on caffeine-induced Ca2+ release in vascular smooth muscle and that cellular Ca2+ levels may affect the action of ryanodine.
Topics: Alkaloids; Animals; Aorta, Thoracic; Caffeine; Calcium; Guinea Pigs; In Vitro Techniques; Male; Muscle Contraction; Muscle, Smooth, Vascular; Rats; Ryanodine
PubMed: 2615045
DOI: 10.1254/jjp.51.531 -
Heart Rhythm May 2018Aging is associated with an increased incidence of atrioventricular nodal (AVN) dysfunction.
Structural and functional remodeling of the atrioventricular node with aging in rats: The role of hyperpolarization-activated cyclic nucleotide-gated and ryanodine 2 channels.
BACKGROUND
Aging is associated with an increased incidence of atrioventricular nodal (AVN) dysfunction.
OBJECTIVE
The aim of this study was to investigate the structural and functional remodeling in the atrioventricular junction (AVJ) with aging.
METHODS
Electrophysiology, histology, and immunohistochemistry experiments on male Wistar Hannover rats aged 3 months (n = 24) and 2 years (n = 15) were performed. Atrio-His (AH) interval, Wenkebach cycle length (WBCL), and AVN effective refractory period (AVNERP) were measured. Cesium (2 mM) was used to block hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, while ryanodine (2 μM) was used to block ryanodine 2 (RyR2) channels. Protein expression from different regions of the AVJ was studied using immunofluorescence. The expression of connexins (connexin 43 and connexin 40), ion channels (Hyperpolarization-activated cyclic nucleotide-gated channel 4 (HCN4), voltage sensitive sodium channel (Na1.5), and L-Type calcium channel (Ca1.3)), and calcium handling proteins (RyR2 and sarco/endoplasmic reticulum calcium ATPaset type 2a (SERCA2a)) were measured. Morphological characteristics were studied with histology.
RESULTS
Without drugs to block HCN and RyR2 channels, there was prolongation of the AH interval, WBCL, and AVNERP (P < .05) with aging. In young rats only, cesium prolonged the AH interval, WBCL, and AVNERP (P < .01). Ryanodine prolonged the AH interval and WBCL (P < .01) in both young and old rats. Immunofluorescence revealed that with aging, connexin 43, HCN4, Na1.5, and RyR2 downregulate in the regions of the AVJ and connexin 40, SERCA2a, and Ca1.3 upregulate (P < .05). Aging results in cellular hypertrophy, loosely packed cells, a decrease in the number of nuclei, and an increase in collagen content.
CONCLUSION
Heterogeneous ion channel expression changes were observed in the AVJ with aging. For the first time, we have shown that HCN and RyR2 play an important role in AVN dysfunction with aging.
Topics: Aging; Animals; Atrioventricular Node; Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; Immunohistochemistry; Male; Models, Animal; Patch-Clamp Techniques; Rats; Rats, Wistar; Ryanodine; Ryanodine Receptor Calcium Release Channel
PubMed: 29288034
DOI: 10.1016/j.hrthm.2017.12.027 -
The Journal of Biological Chemistry Nov 2020Structural analyses identified the central domain of ryanodine receptor (RyR) as a transducer converting conformational changes in the cytoplasmic platform to the RyR...
Structural analyses identified the central domain of ryanodine receptor (RyR) as a transducer converting conformational changes in the cytoplasmic platform to the RyR gate. The central domain is also a regulatory hub encompassing the Ca-, ATP-, and caffeine-binding sites. However, the role of the central domain in RyR activation and regulation has yet to be defined. Here, we mutated five residues that form the Ca activation site and 10 residues with negatively charged or oxygen-containing side chains near the Ca activation site. We also generated eight disease-associated mutations within the central domain of RyR2. We determined the effect of these mutations on Ca, ATP, and caffeine activation and Mg inhibition of RyR2. Mutating the Ca activation site markedly reduced the sensitivity of RyR2 to Ca and caffeine activation. Unexpectedly, Ca activation site mutation E3848A substantially enhanced the Ca-independent basal activity of RyR2, suggesting that E3848A may also affect the stability of the closed state of RyR2. Mutations in the Ca activation site also abolished the effect of ATP/caffeine on the Ca-independent basal activity, suggesting that the Ca activation site is also a critical determinant of ATP/caffeine action. Mutating residues with negatively charged or oxygen-containing side chains near the Ca activation site significantly altered Ca and caffeine activation and reduced Mg inhibition. Furthermore, disease-associated RyR2 mutations within the central domain significantly enhanced Ca and caffeine activation and reduced Mg inhibition. Our data demonstrate that the central domain plays an important role in channel activation, channel regulation, and closed state stability.
Topics: Adenosine Triphosphate; Binding Sites; Caffeine; Calcium; Calcium Signaling; HEK293 Cells; Humans; Magnesium; Molecular Dynamics Simulation; Mutagenesis, Site-Directed; Myocardium; Protein Binding; Protein Stability; Protein Structure, Tertiary; Ryanodine; Ryanodine Receptor Calcium Release Channel
PubMed: 32878990
DOI: 10.1074/jbc.RA120.013512 -
The Journal of Biological Chemistry Jan 1989Using density gradient centrifugation and [3H]ryanodine as a specific marker, the ryanodine receptor-Ca2+ release channel complex from Chaps-solubilized canine cardiac...
Using density gradient centrifugation and [3H]ryanodine as a specific marker, the ryanodine receptor-Ca2+ release channel complex from Chaps-solubilized canine cardiac sarcoplasmic reticulum (SR) has been purified in the form of an approximately 30 S complex, comprised of Mr approximately 400,000 polypeptides. Purification resulted in a specific activity of approximately 450 pmol bound ryanodine/mg of protein, a 60-70% recovery of ryanodine binding activity, and retention of the high affinity ryanodine binding site (KD = 3 nM). Negative stain electron microscopy revealed a 4-fold symmetric, four-leaf clover structure, which could fill a box approximately 30 x 30 nm and was thus morphologically similar to the SR-transverse-tubule, junctionally associated foot structure. The structural, sedimentation, and ryanodine binding data strongly suggest there is one high affinity ryanodine binding site/30 S complex, comprised of four Mr approximately 400,000 subunits. Upon reconstitution into planar lipid bilayers, the purified complex exhibited a Ca2+ conductance (70 pS in 50 mM Ca2+) similar to that of the native cardiac Ca2+ release channel (75 pS). The reconstituted complex was also found to conduct Na+ (550 pS in 500 mM Na+) and often to display complex Na+ subconducting states. The purified channel could be activated by micromolar Ca2+ or millimolar ATP, inhibited by millimolar Mg2+ or micromolar ruthenium red, and modified to a long-lived open subconducting state by ryanodine. The sedimentation, subunit composition, morphological, and ryanodine binding characteristics of the purified cardiac ryanodine receptor-Ca2+ release channel complex were similar to those previously described for the purified ryanodine receptor-Ca2+ release channel complex from fast-twitch skeletal muscle.
Topics: Animals; Calcium; Dogs; Heart Ventricles; Ion Channels; Kinetics; Lipid Bilayers; Molecular Weight; Muscles; Myocardium; Organ Specificity; Receptors, Cholinergic; Ryanodine; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum
PubMed: 2463249
DOI: No ID Found -
The Journal of Urology Feb 1997To investigate the effect of independently inhibiting calcium influx from extracellular sources and calcium release from intracellular stores on the ability of the...
PURPOSE
To investigate the effect of independently inhibiting calcium influx from extracellular sources and calcium release from intracellular stores on the ability of the urinary bladder to generate pressure and empty.
MATERIALS AND METHODS
Rabbit bladders were mounted in an in vitro whole organ bath and filled with 15 ml. saline. Each bladder was incubated separately in Tyrode's solution, with diltiazem (10 microM), to block extracellular calcium influx, or with thapsigargin (40 microM) and ryanodine (80 microM), to block the uptake and release of calcium from the sarcoplasmic reticulum. The bladder was then stimulated isometrically with field stimulation (32 Hz), and to empty with field stimulation and with bethanechol (250 microM), independently. During stimulation, transmural pressure and volume emptied were measured. From these, flow rate, power, and external mechanical work were calculated.
RESULTS
In the presence of diltiazem, the time to maximal pressure decreased while the rate of pressure generation increased. This results from increased participation of intracellular calcium release, which occurs rapidly and near the smooth muscle filaments, decreasing the diffusion time. In the presence of thapsigargin and ryanodine the maximal rate of pressure generation was decreased, due to the increased diffusion time required for calcium to move to the muscle filaments from extracellular sites.
CONCLUSIONS
The current study demonstrates that bladder pressure generation and emptying are dependent upon both an influx of calcium through L-type calcium channels (inhibited by diltiazem) and the stimulated release of calcium from the SR (inhibited by thapsigargin and ryanodine).
Topics: Animals; Bethanechol; Calcium; Diltiazem; Electric Stimulation; Enzyme Inhibitors; In Vitro Techniques; Parasympathomimetics; Pressure; Rabbits; Ryanodine; Thapsigargin; Urinary Bladder
PubMed: 8996408
DOI: No ID Found -
Acta Biochimica Polonica Nov 2019The aim of this study was to determine a match between DNA recovered from evidence material, such as knocked down red deer, and from comparative material in form of two...
The aim of this study was to determine a match between DNA recovered from evidence material, such as knocked down red deer, and from comparative material in form of two brown traces on the bonnet of a car driven by a person suspected of knocking down the animal. The spots coming from the car provided no DNA profile, which questioned that they originated from a red deer and ruled out performance of a comparative DNA analysis. For this reason, the material obtained from the blood smear was analyzed for species identification. The method applied can discriminate between cattle, red deer and roe deer based on restriction analysis (Tsp509I) of PCR product (195bp), obtained by amplifying a fragment of the cytochrome b coding gene. Because the obtained restriction profile confirmed the match with red deer DNA for one trace, and in the second case ruled out that the biological traces originated from the species mentioned above, the PCR products were subjected to sequencing. In both cases, 195bp PCR products that were 98% homologous with red deer DNA sequence-NC_007704.2-trace1 and with the gene coding for the human ryanodine receptor-NC_008799.2-trace2. The quantity and quality of DNA obtained from the traces collected from the car bonnet did not allow confirmation of the involvement of a specific animal in the event, but the applied method made it possible to determine the species from which the obtained traces originated. Furthermore, the applied method, which was used earlier to determine cervine DNA, was successfully used to detect human DNA.
Topics: Accidents, Traffic; Animals; Cattle; Cytochromes b; DNA; Deer; Forensic Genetics; Humans; Polymorphism, Genetic; Ryanodine; Species Specificity
PubMed: 31703163
DOI: 10.18388/abp.2019_2818 -
Molecular and Biochemical Parasitology May 2010Cholinergic anthelmintics (like levamisole) are important drugs but resistance with reduced responses by the parasite to these compounds is a concern. There is a need to...
Cholinergic anthelmintics (like levamisole) are important drugs but resistance with reduced responses by the parasite to these compounds is a concern. There is a need to study and understand mechanisms that affect the amplitude of the responses of parasites to these drugs. In this paper, we study interactions of levamisole and ryanodine receptors on contractions of Ascaris suum body muscle flaps. In our second paper, we extend these observations to examine electrophysiological interactions of levamisole, ryanodine receptors (RyRs) and AF2. We report that the maximum force of contraction, g(max), was dependent on the extracellular concentration of calcium but the levamisole EC(50) (0.8 microM) was not. The relationship between maximum force of contraction and extracellular calcium was described by the Michaelis-Menten equation with a K(m) of 1.8mM. Ryanodine inhibited g(max) without effect on EC(50); ryanodine inhibited only 44% of the maximum contraction (K(i) of 40 nM), revealing a ryanodine-insensitive component in the levamisole excitation-contraction pathway. Dantrolene had the same effect as ryanodine but was less potent. The neuropeptide AF2 (1 microM) decreased the levamisole EC(50) to 0.2 microM without effect on g(max); 0.1 microM ryanodine and 100 microM dantrolene, inhibited the g(max) of the AF2-potentiated levamisole response. High concentrations of caffeine, 30 mM, produced weak contraction of the body-flap preparation. Caffeine behaved like ryanodine in that it inhibited the maximum force of contraction, g(max), without effects on the levamisole EC(50). Thus, RyRs play a modulatory role in the levamisole excitation-contraction pathway by affecting the maximum force of contraction without an effect on levamisole EC(50). The levamisole excitation-contraction coupling is graded and has at least two pathways: one sensitive to ryanodine and one not.
Topics: Animals; Anthelmintics; Ascaris suum; Caffeine; Calcium; Cholinergic Agonists; Dantrolene; Furylfuramide; Levamisole; Muscle Contraction; Ryanodine; Ryanodine Receptor Calcium Release Channel
PubMed: 20064566
DOI: 10.1016/j.molbiopara.2009.12.007