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The Journal of Experimental Biology Jun 2006Reactive oxygen species and related oxidative damage have been implicated in the initiation of acute pancreatitis, a disease characterised in its earliest stages by...
Reactive oxygen species and related oxidative damage have been implicated in the initiation of acute pancreatitis, a disease characterised in its earliest stages by disruption of intracellular Ca2+ homeostasis. The present study was carried out in order to establish the effect of the organic pro-oxidant, tert-butylhydroperoxide (tBHP), on the mobilisation of intracellular Ca2+ stores in isolated rat pancreatic acinar cells and the mechanisms underlying this effect. Cytosolic free Ca2+ concentrations ([Ca2+]c) were monitored using a digital microspectrofluorimetric system in fura-2 loaded cells. In the presence of normal extracellular Ca2+ concentrations ([Ca2+]o), perfusion of pancreatic acinar cells with 1 mmol l-1 tBHP caused a slow sustained increase in [Ca2+]c. This increase was also observed in a nominally Ca2+-free medium, indicating a release of Ca2+ from intracellular stores. Pretreatment of cells with tBHP abolished the typical Ca2+ response of both the physiological agonist CCK-8 (1 nmol l-1) and thapsigargin (TPS, 1 micromol l-1), an inhibitor of the SERCA pump, in the absence of extracellular Ca2+. Similar results were observed with carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP, 0.5 micromol l-1), a mitochondrial uncoupler. In addition, depletion of either agonist-sensitive Ca2+ pools by CCK-8 or TPS or mitochondrial Ca2+ pools by FCCP were unable to prevent the tBHP-induced Ca2+ release. By contrast, simultaneous administration of TPS and FCCP clearly abolished the tBHP-induced Ca2+ release. These results show that tBHP releases Ca2+ from agonist-sensitive intracellular stores and from mitochondria. On the other hand, simultaneous application of FCCP and of 2-aminoethoxydiphenylborane (2-APB), a blocker of IP3-mediated Ca2+ release, was unable to suppress the increase in [Ca2+]c induced by tBHP, while the application of 50 micromol l-1 of ryanodine (which is able to block the ryanodine channels) inhibits tBHP-evoked Ca2+ mobilisation. These findings indicate that tBHP releases Ca2+ from non-mitochondrial Ca2+ pools through ryanodine channels.
Topics: Animals; Calcium; Calcium Channel Blockers; Calcium Channels; Calcium Signaling; Cells, Cultured; Inositol 1,4,5-Trisphosphate; Male; Pancreas; Rats; Rats, Wistar; Ryanodine; tert-Butylhydroperoxide
PubMed: 16709917
DOI: 10.1242/jeb.02250 -
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 -
European Journal of Pharmacology Jun 1988In rat ventricular muscles, ryanodine (10-30 nM) evoked tension oscillation in the relaxation phase of an isometric twitch (relaxation oscillations) and an increase of...
In rat ventricular muscles, ryanodine (10-30 nM) evoked tension oscillation in the relaxation phase of an isometric twitch (relaxation oscillations) and an increase of tonic tension (ryanodine contracture). Both events were more pronounced in Ca2+-loaded rat muscles due to the addition of 0.5 microM adrenaline, an increase in the Ca2+ concentration in the solution and high muscle activity. The ryanodine-induced tension oscillations were comparable to those triggered by caffeine in this species. In both cases, blockers of the release of Ca2+ from the sarcoplasmic reticulum, namely tetracaine and dantrolene, abolished the relaxation oscillations and the ryanodine contracture. The results suggest that the ability of low concentrations of ryanodine to facilitate the release of Ca2+ from the sarcoplasmic reticulum, as shown recently in biochemical experiments, makes a direct contribution to triggering the relaxation oscillations and the ryanodine contracture in intact ventricular muscles. The Ca2+ load of the sarcoplasmic reticulum appears to be essential for the manifestation of the ryanodine Ca2+-releasing activity in rat ventricular muscles.
Topics: Alkaloids; Animals; Caffeine; Calcium; Dantrolene; In Vitro Techniques; Isometric Contraction; Myocardial Contraction; Papillary Muscles; Rats; Rats, Inbred Strains; Ryanodine; Tetracaine
PubMed: 3416914
DOI: 10.1016/0014-2999(88)90015-5 -
The Journal of General Physiology Sep 2022Flecainide, a cardiac class 1C blocker of the surface membrane sodium channel (NaV1.5), has also been reported to reduce cardiac ryanodine receptor (RyR2)-mediated...
Flecainide, a cardiac class 1C blocker of the surface membrane sodium channel (NaV1.5), has also been reported to reduce cardiac ryanodine receptor (RyR2)-mediated sarcoplasmic reticulum (SR) Ca2+ release. It has been introduced as a clinical antiarrhythmic agent for catecholaminergic polymorphic ventricular tachycardia (CPVT), a condition most commonly associated with gain-of-function RyR2 mutations. Current debate concerns both cellular mechanisms of its antiarrhythmic action and molecular mechanisms of its RyR2 actions. At the cellular level, it targets NaV1.5, RyR2, Na+/Ca2+ exchange (NCX), and additional proteins involved in excitation-contraction (EC) coupling and potentially contribute to the CPVT phenotype. This Viewpoint primarily addresses the various direct molecular actions of flecainide on isolated RyR2 channels in artificial lipid bilayers. Such studies demonstrate different, multifarious, flecainide binding sites on RyR2, with voltage-dependent binding in the channel pore or voltage-independent binding at distant peripheral sites. In contrast to its single NaV1.5 pore binding site, flecainide may bind to at least four separate inhibitory sites on RyR2 and one activation site. None of these binding sites have been specifically located in the linear RyR2 sequence or high-resolution structure. Furthermore, it is not clear which of the inhibitory sites contribute to flecainide's reduction of spontaneous Ca2+ release in cellular studies. A confounding observation is that flecainide binding to voltage-dependent inhibition sites reduces cation fluxes in a direction opposite to physiological Ca2+ flow from SR lumen to cytosol. This may suggest that, rather than directly blocking Ca2+ efflux, flecainide can reduce Ca2+ efflux by blocking counter currents through the pore which otherwise limit SR membrane potential change during systolic Ca2+ efflux. In summary, the antiarrhythmic effects of flecainide in CPVT seem to involve multiple components of EC coupling and multiple actions on RyR2. Their clarification may identify novel specific drug targets and facilitate flecainide's clinical utilization in CPVT.
Topics: Anti-Arrhythmia Agents; Calcium; Flecainide; Humans; Myocytes, Cardiac; Ryanodine; Ryanodine Receptor Calcium Release Channel; Sodium; Tachycardia, Ventricular
PubMed: 35713932
DOI: 10.1085/jgp.202213089 -
Journal of Neurophysiology Sep 20026-((4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)amino)hexanoic acid ryanodine (BODIPY-ryanodine) binding and Ca(2+) imaging were used to study...
6-((4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)amino)hexanoic acid ryanodine (BODIPY-ryanodine) binding and Ca(2+) imaging were used to study the properties of ryanodine receptors (RyRs) and cytoplasmic Ca(2+) (Ca) changes in neurons cultured from the embryonic rat hippocampus during the earliest stages of differentiation. Baseline Ca levels declined from 164 +/- 5 (SD) nM at early stages to 70 +/- 4 nM in differentiated neurons. Fluorescent BODIPY-ryanodine binding signals identified activated RyRs in somata, which were eliminated by removal of external Ca(2+) or by blockage of Ca(2+) entry through L-type but not N-type Ca(2+) channels. The GABA synthesis inhibitor 3-mercaptopropionic acid completely abolished ryanodine binding. Caffeine or K(+)-depolarization inhibited the activity of RyRs at very early stages of differentiation but had stimulatory effects at later stages after a network of processes had formed. BayK-8644 stimulated RyRs throughout all regions of all differentiating cells. The results suggest that in differentiating embryonic hippocampal neurons the activity of RyRs is maintained via Ca(2+) entering through L-type Ca(2+) channels. The mode of activation of L-type voltage-gated Ca(2+) channels with either membrane depolarization or specific pharmacological agents affects the coupled activity of RyRs differently as neurons differentiate processes and networks.
Topics: 3-Mercaptopropionic Acid; 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Animals; Caffeine; Calcium; Calcium Channel Agonists; Calcium Channels, L-Type; Cell Differentiation; Cells, Cultured; Hippocampus; Neurons; Potassium Chloride; Rats; Rats, Sprague-Dawley; Ryanodine; Ryanodine Receptor Calcium Release Channel
PubMed: 12205130
DOI: 10.1152/jn.2002.88.3.1077 -
British Journal of Pharmacology Oct 19901. Action potentials from guinea-pig single ventricular myocytes were interrupted by application of a 300 ms voltage clamp to -40 mV in order to evoke the Ca-activated...
1. Action potentials from guinea-pig single ventricular myocytes were interrupted by application of a 300 ms voltage clamp to -40 mV in order to evoke the Ca-activated tail current which is thought to be carried by Na:Ca exchange. Stimulation frequency was 1 Hz and temperature 36 degrees C. 2. The actions of ryanodine (1 microM and 10 microM) and caffeine (1 mM and 10 mM) on Ca-activated tail currents were investigated. 3. Exposure to 10 mM caffeine and ryanodine reduced tail currents associated with very abbreviated (12 ms duration) action potentials and greatly reduced the difference between first and steady-state tail currents at this action potential duration. These observations were interpreted in terms of suppression of Ca release from the sarcoplasmic reticulum (SR) stores. 4. Tail current decay during the voltage clamp is thought to reflect the fall in [Ca]i which accompanies muscle relaxation. Current decay is dependent on Ca extrusion via Na:Ca exchange and on Ca accumulation by the SR stores. Time constants of tail current decay were seen to decrease with increasing action potential duration. This relationship was not affected by 1 mM caffeine or 1 microM ryanodine. Ryanodine at 10 microM and 10 mM caffeine abolished this relationship and increased the time constants of current decay. An increase in the time constant of tail current decay was thought to reflect a reduction in the rate of Ca accumulation by the sarcoplasmic reticulum. 5. The actions of caffeine and ryanodine on the Ca-activated tail currents are consistent with a dose-dependent leakage of Ca from the SR Ca stores. The Ca-activated tail current appears to be a useful tool in the study of Ca homeostasis.
Topics: Action Potentials; Animals; Caffeine; Calcium; Guinea Pigs; Heart; Heart Ventricles; In Vitro Techniques; Membrane Potentials; Ryanodine
PubMed: 2257440
DOI: 10.1111/j.1476-5381.1990.tb12721.x -
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 -
Zhonghua Xin Xue Guan Bing Za Zhi Sep 2011To observe the effects of ryanodine on rapamycin treated endothelial outgrowth cells (EOCs).
OBJECTIVE
To observe the effects of ryanodine on rapamycin treated endothelial outgrowth cells (EOCs).
METHODS
The mononuclear cells were harvested from umbilical cord blood by Ficoll density gradient centrifugation, then induced into EOCs and expanded in vitro. The endothelial characteristics of EOCs were identified by immunostaining and fluorescent staining. The EOCs were pretreated with or without ryanodine (10 µmol/L) for 1 h, and then treated with or without rapamycin (10 nmol/L) for 24 h. Proliferation was evaluated by CCK8 and migration was measured by Transwell. The protein expression of EOCs was evaluated by immunobloting technique with total eNOS antibody and phospho-eNOS (Thr495) antibody.
RESULTS
Compared with control group, the proliferation and migration capacities of EOCs were significantly reduced while the phosphorylation of eNOS (Thr495) protein was significantly upregulated in rapamycin group (P < 0.05), expression of total eNOS was not affected by rapamycin (P > 0.05). Compared with rapamycin group, the proliferation and migration capacities of EOCs were significantly increased and the phosphorylation of eNOS (Thr495) protein was significantly downregulated in ryanodine + rapamycin group (P < 0.05). The proliferation and migration capacities, the phosphorylation of eNOS (Thr495) protein and the expression of total eNOS were not affected by ryanodine alone (P > 0.05).
CONCLUSIONS
Rapamycin reduced proliferation and migration capacities while upregulated the phosphorylation of eNOS (Thr495) protein of EOCs and these effects could be partly reversed by cotreatment with ryanodine.
Topics: Cells, Cultured; Down-Regulation; Drug Synergism; Endothelial Cells; Humans; Nitric Oxide Synthase Type III; Phosphorylation; Ryanodine; Sirolimus
PubMed: 22321235
DOI: No ID Found -
The Biochemical Journal Sep 1997Dantrolene inhibits and ryanodine stimulates calcium release from skeletal-muscle sarcoplasmic reticulum (SR), the former by an unknown mechanism, and the latter by...
Dantrolene inhibits and ryanodine stimulates calcium release from skeletal-muscle sarcoplasmic reticulum (SR), the former by an unknown mechanism, and the latter by activating the ryanodine receptor (RyR), the primary Ca2+-release channel of SR. Dantrolene is used to treat malignant hyperthermia (MH), a genetic predisposition to excessive intracellular Ca2+ release upon exposure to volatile anaesthetics. Porcine MH results from a point mutation in the SR RyR that alters the open probability of the channel, and is reflected in altered [3H]ryanodine binding parameters. Specific binding sites for [3H]dantrolene and [3H]ryanodine co-distribute on SR that has been isolated by discontinuous sucrose gradient centrifugation. If the two drug-binding sites are functionally linked, [3H]dantrolene binding might be affected both by pharmacological and by genetic modulators of the functional state of the RyR. Accordingly, we compared the characteristics of [3H]dantrolene binding to porcine malignant-hyperthermia-susceptible and normal-skeletal-muscle SR, and examined the effects of RyR modulators on [3H]dantrolene binding to these membranes. Additionally, the feasibility of separating the SR binding sites for [3H]dantrolene and [3H]ryanodine was investigated. No significant differences in [3H]dantrolene binding characteristics to SR membranes from the two muscle types were detected, and the Bmax ratio for [3H]dantrolene/[3H]ryanodine was 1.4(+/-0.1):1 in both muscle types. [3H]Dantrolene binding is unaffected by the RyR modulators caffeine, ryanodine, Ruthenium Red and calmodulin, and neither dantrolene nor azumolene have any effect on [3H]ryanodine binding. Additionally, distinct peaks of [3H]dantrolene and [3H]ryanodine binding are detected in SR membranes fractionated by linear sucrose centrifugation, although no differences in protein patterns are detected by SDS/PAGE or Western-blot analysis. We suggest that the binding sites for these two drugs are pharmacologically distinct, and may exist on separate molecules.
Topics: Animals; Binding Sites; Cell Membrane; Dantrolene; Malignant Hyperthermia; Muscle Relaxants, Central; Muscle, Skeletal; Osmolar Concentration; Radioligand Assay; Ryanodine; Swine
PubMed: 9307036
DOI: 10.1042/bj3260847 -
Science (New York, N.Y.) Aug 2016(+)-Ryanodine and (+)-ryanodol are complex diterpenoids that modulate intracellular calcium-ion release at ryanodine receptors, ion channels critical for skeletal and...
(+)-Ryanodine and (+)-ryanodol are complex diterpenoids that modulate intracellular calcium-ion release at ryanodine receptors, ion channels critical for skeletal and cardiac muscle excitation-contraction coupling and synaptic transmission. Chemical derivatization of these diterpenoids has demonstrated that certain peripheral structural modifications can alter binding affinity and selectivity among ryanodine receptor isoforms. Here, we report a short chemical synthesis of (+)-ryanodol that proceeds in only 15 steps from the commercially available terpene (S)-pulegone. The efficiency of the synthesis derives from the use of a Pauson-Khand reaction to rapidly build the carbon framework and a SeO2-mediated oxidation to install three oxygen atoms in a single step. This work highlights how strategic C-O bond constructions can streamline the synthesis of polyhydroxylated terpenes by minimizing protecting group and redox adjustments.
Topics: Biological Products; Cyclohexane Monoterpenes; Monoterpenes; Oxidation-Reduction; Oxygen; Ryanodine; Ryanodine Receptor Calcium Release Channel; Selenium Oxides
PubMed: 27563092
DOI: 10.1126/science.aag1028