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The Journal of Physiology 19911. Charge movement and myoplasmic calcium transients were simultaneously recorded from frog skeletal muscle fibres by using the double-seal Vaseline-gap technique.... (Comparative Study)
Comparative Study
1. Charge movement and myoplasmic calcium transients were simultaneously recorded from frog skeletal muscle fibres by using the double-seal Vaseline-gap technique. Calcium transients were monitored with the fluorescent indicator Rhod-2. 2. Ryanodine modified the kinetics and the total amount of charge moved during depolarizing pulses (Q(on)), while it did not significantly modify the charge after repolarization (Q(off)). The extracellular application of 100 microM-ryanodine elicited a temporary initial increase of the delayed component of charge movement (Q gamma) and the calcium transient. Both phenomena were later blocked with the same temporal course and to the same extent. 3. The blockade of Q gamma and the calcium transient was also observed with ryanodine concentrations of 1-10 microM. For membrane potentials positive to -10mV, the Qon measured was larger in the presence of ryanodine; Qoff was not modified. 4. Tetracaine (400-500 microM) blocked a similar delayed component of Qon, identified as Q gamma, as well as the calcium transient monitored simultaneously. This effect was observed in the first minutes after the addition of tetracaine to the extracellular solution. 5. Tetracaine blocked a faster initial component of Qon for voltages positive to -10 mV, corresponding to the voltage range of activation of the calcium current. At these same membrane potentials, Qoff was also reduced to a similar extent to Qon. 6. Ryanodine and tetracaine showed different effects on calcium current. Whereas the slow calcium current was not modified upon the addition of ryanodine, it was completely blocked in the presence of tetracaine. The blockade of the slow calcium current made evident the fast calcium current. The effects of tetracaine on the charge movement, the calcium transient and the slow calcium current were reversible. 7. These results suggest that ryanodine and tetracaine may act at different sites. Ryanodine exerts its effect on the sarcoplasmic reticulum ryanodine receptor, blocking calcium release and Q gamma, while tetracaine at these concentrations may affect the release channel and the dihydropyridine receptor, causing a blockade of the charge movement, calcium transient and calcium current.
Topics: Animals; Calcium; Calcium Channels; Electrophysiology; In Vitro Techniques; Ion Channel Gating; Muscles; Rana pipiens; Ryanodine; Tetracaine; Time Factors
PubMed: 1666652
DOI: 10.1113/jphysiol.1991.sp018715 -
Circulation Research Nov 1989Extent, time course, and underlying mechanisms of the negative inotropic effect of ryanodine were examined in 22 length-clamped ferret right ventricular papillary... (Comparative Study)
Comparative Study
Extent, time course, and underlying mechanisms of the negative inotropic effect of ryanodine were examined in 22 length-clamped ferret right ventricular papillary muscles paced 12/min at 25 degrees C. After 60 minutes of exposure to 5 microM ryanodine a new steady state was attained with developed forces averaging 10-15% of maximum twitch force. Ryanodine does not pharmacologically excise the sarcoplasmic reticulum (SR) in this preparation. Ryanodine does not appreciably inhibit the ability of the SR to take up Ca2+ as evidenced by the potentiated beats obtained after a short pause that are nearly as large after ryanodine as before. On comparing equipotent beats before and after ryanodine, we found that ryanodine actually increases the rate at which Ca2+ is released during the twitch if the SR Ca2+ stores are equal or similar. The evidence for this conclusion is a larger maximum rate of tension rise and briefer time to peak tension after ryanodine. Since ryanodine increases the time that SR Ca2+ release channels are open and decreases their conductivity, it must follow that the former effect predominates over the latter in our experiments. Ryanodine increases the leakiness of the SR during diastole probably by inhibiting closure of SR Ca2+ release channels. The evidence for this conclusion is as follows: the early peak of the restitution curves after ryanodine, the brevity of the time required for a rested state contraction after ryanodine, and the small amplitude of the steady-state contraction at a rate of 12/min. The SR leaks even in the absence of ryanodine, but if external Ca2+ is so high that Ca2+ loss from the cell is slowed or a Ca2+ leak into the cell through the sarcolemma cancels the SR leak, then the effects of the SR leak are minimized. The evidence for this conclusion is the time required for rested-state contraction to occur or the slope of the descending limb of restitution curve; however, in presence of ryanodine even high external Ca2+ cannot prevent rapid depletion of SR Ca2+ stores. Even though we have presented evidence for a mechanism whereby ryanodine increases the number of open SR Ca2+ release channels in both systole and diastole, we do not mean to imply that most of them stay open in diastole; the SR would leak too fast to accumulate any Ca2+ for the potentiated beat. Thus, probably most channels close after being open a certain length of time, even in the presence of ryanodine.
Topics: Alkaloids; Animals; Calcium; Cardiac Pacing, Artificial; Ferrets; Homeostasis; In Vitro Techniques; Myocardial Contraction; Osmolar Concentration; Papillary Muscles; Ryanodine; Time Factors
PubMed: 2805244
DOI: 10.1161/01.res.65.5.1270 -
Urological Research 1995Norepinephrine release from adrenergic nerve terminals leads to a rise in intracellular Ca2+, which promotes penile smooth muscle contraction and detumescence. Ca2+...
Norepinephrine release from adrenergic nerve terminals leads to a rise in intracellular Ca2+, which promotes penile smooth muscle contraction and detumescence. Ca2+ sources are the extracellular space and sarcoplasmic Ca2+ stores. To elucidate the role of intracellular stores strips from rabbit erectile tissue were investigated in an organ bath study. Contractions were elicited by phenylephrine (PE) and electrical stimulation. Incubation in Ca(2+)-free solution as well as exposure to nifedipine did not abolish electrical or PE-induced contraction. Ryanodine (10(-5) mol/l), a functional blocker of sarcoplasmic Ca2+ channels, significantly reduced PE response. In the presence of caffeine (10(-3) mol/l) the effect was significantly enhanced. Addition of nifedipine nearly abolished the contraction. These results provide evidence for intracellular Ca2+ pools in cavernosal tissue and indicate that the alpha 1-adrenoceptor-induced contraction requires the opening of voltage-gated Ca2+ channels and the release of Ca2+ from intracellular stores.
Topics: Animals; Caffeine; Calcium; Electric Stimulation; In Vitro Techniques; Intracellular Membranes; Male; Nifedipine; Penile Erection; Penis; Phenylephrine; Potassium; Rabbits; Ryanodine; Solutions
PubMed: 7740661
DOI: 10.1007/BF00296883 -
Biophysical Journal Aug 1996We purified and characterized ryanotoxin, an approximately 11.4-kDa peptide from the venom of the scorpion Buthotus judiacus that induces changes in ryanodine receptors...
We purified and characterized ryanotoxin, an approximately 11.4-kDa peptide from the venom of the scorpion Buthotus judiacus that induces changes in ryanodine receptors of rabbit skeletal muscle sarcoplasmic reticulum analogous to those induced by the alkaloid ryanodine. Ryanotoxin stimulated Ca2+ release from sarcoplasmic reticulum vesicles and induced a state of reduce unit conductance with a mean duration longer than that of unmodified ryanodine receptor channels. With Cs+ as the current carrier, the slope conductance of the state induced by 1 microM ryanotoxin was 163 +/- 12 pS, that of the state induced by 1 microM ryanodine was 173 +/- 26 pS, and that of control channels was 2.3-fold larger (396 +/- 25 pS). The distribution of substate events induced by 1 microM RyTx was biexponential and was fitted with time constants approximately 10 times shorter than those fitted to the distribution of substates induced by 1 microM ryanodine. Bath-applied 5 microM ryanotoxin had no effect on the excitability of mouse myotubes in culture. When 5 microM ryanotoxin was dialyzed into the cell through the patch pipette in the whole-cell configuration, there was a voltage-dependent increase in the amplitude of intracellular Ca2+ transients elicited by depolarizing potentials in the range of -30 to +50 mV. Ryanotoxin increased the binding affinity of [3H]ryanodine in a reversible manner with a 50% effective dose (ED50) of 0.16 microM without altering the maximum number (Bmax) of [3H]ryanodine-binding sites. This result suggested that binding sites for ryanotoxin and ryanodine were different. Ryanotoxin should prove useful in identifying domains coupling the ryanodine receptor to the voltage sensor, or domains affecting the gating and conductance of the ryanodine receptor channel.
Topics: Animals; Calcium; Calcium Channels; Cells, Cultured; Cesium; Chromatography, High Pressure Liquid; Electric Conductivity; Fetus; Kinetics; Membrane Potentials; Mice; Muscle Proteins; Muscle, Skeletal; Rabbits; Ruthenium Red; Ryanodine; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum; Scorpion Venoms
PubMed: 8842209
DOI: 10.1016/S0006-3495(96)79270-6 -
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 -
Journal of Medicinal Chemistry Apr 1987The topography and toxicological relevance of the Ca2+-ryanodine receptor complex are evaluated with ryanodine and two natural analogues (9,21-didehydro and the new... (Comparative Study)
Comparative Study
The topography and toxicological relevance of the Ca2+-ryanodine receptor complex are evaluated with ryanodine and two natural analogues (9,21-didehydro and the new 18-hydroxy), 13 ryanoid derivatives (prepared from ryanodine and didehydroryanodine by functionalizing the available pyrrole, olefin, and hydroxyl substituents), and four degradation products. The potency of ryanoids at the skeletal muscle sarcoplasmic reticulum specific binding site generally parallels their toxicity to mice, supporting the toxicological relevance of the Ca2+-ryanodine receptor. The optimal receptor potency of ryanodine and didehydroryanodine is reduced 3-14-fold by hydroxylation at an isopropyl methyl substituent, epimerization at C9, oxidation or acetylation of the C10-hydroxyl, or epoxidation at the 9,21-position; other ryanoids are less active. Ryanodol and didehydroryanodol, in contrast to ryanodine and didehydroryanodine, have low toxicity to mice and little activity at the mammalian receptor, yet they are potent knockdown agents for injected houseflies or cockroaches, suggesting a possible difference in the target sites of mammals and insects.
Topics: Alkaloids; Alkylation; Animals; Calcium; Calcium Channel Blockers; Diptera; Insecticides; Ion Channels; Male; Mice; Molecular Conformation; Muscle Contraction; Rabbits; Receptors, Cholinergic; Ryanodine; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum; Seizures; Species Specificity; Structure-Activity Relationship
PubMed: 2435905
DOI: 10.1021/jm00387a022 -
Brain Research Feb 1991High affinity [3H]ryanodine binding sites were characterized in P1 (crude nuclear), P2 (mitochondrial/synaptosomal) and P3 (microsomal) subcellular fractions of rat...
High affinity [3H]ryanodine binding sites were characterized in P1 (crude nuclear), P2 (mitochondrial/synaptosomal) and P3 (microsomal) subcellular fractions of rat brain. Binding in each of the fractions was highest at 37 degrees C and pH 8-9, optimal in the presence of 100 microM Ca2+, 550 microM ATP and 1.0 M KCl, and increased linearly as a function of protein. Saturation analyses revealed a single class of binding sites with mean KD values (nM) of 8.9, 1.6 and 5.7 and Bmax values (fmol/mg protein) of 122, 69 and 106 for the P1, P2 and P3 fractions, respectively. The levels of [3H]ryanodine binding in P1 and P2 fractions of 4 brain regions were fairly uniform while those in P3 fractions were 5-fold greater in cerebral cortex than in the other areas examined. By autoradiography, a high concentration of [3H]ryanodine binding sites was seen in the dentate gyrus and CA3 subregions of the hippocampus. The results suggest that [3H]ryanodine binding sites, perhaps similar to [3H]ryanodine receptors in muscle, are associated with various subcellular structures and are heterogeneously distributed in the CNS.
Topics: Animals; Autoradiography; Binding Sites; Brain; Male; Membranes; Rats; Rats, Inbred Strains; Ryanodine; Subcellular Fractions; Tritium
PubMed: 2054652
DOI: 10.1016/0006-8993(91)91007-n -
The American Journal of Physiology Sep 1997After anesthesia and autonomic blockade, nine dogs chronically instrumented with left ventricular (LV) micromanometers and piezoelectric dimension crystals were studied...
After anesthesia and autonomic blockade, nine dogs chronically instrumented with left ventricular (LV) micromanometers and piezoelectric dimension crystals were studied before and after the intravenous administration of 4 micrograms/kg ryanodine, a specific inhibitor of the sarcoplasmic reticulum Ca2+ release channel. Ryanodine prolonged LV contraction and relaxation (P < 0.001) without changing heart rate, end-diastolic volume (EDV), or end-systolic pressure. Velocity-dependent mechanical parameters were significantly depressed, including the maximal rate of LV pressure rise (dP/dtmax; P < 0.002), the mean velocity of circumferential fiber shortening (P < 0.002), the slope of the dP/dtmax-EDV relation (P < 0.05), and the time constant of LV relaxation (P < 0.01). In contrast, the slopes of the end-systolic pressure-volume (PES-VES) and stroke work (SW)-EDV relations, both force-based parameters, were increased (P < 0.05) or maintained, respectively. Ryanodine reduced overall LV contractile performance, evidenced by significant rightward shifts of the PES-VES, dP/dtmax-EDV, and SW-EDV relations and reduced SW at constant preload (P < 0.02). Thus, in the closed-chest dog, low-dose ryanodine resulted in 1) generalized slowing of LV mechanical events without changes in heart rate or load, 2) dissociation of velocity-based and force-based measures of LV function, with depression of the former but enhancement or maintenance of the latter, and 3) reduced overall LV inotropic performance. These effects are consistent with ryanodine-induced alterations of the Ca2+ transient and altered sarcoplasmic reticulum Ca2+ availability.
Topics: Animals; Diastole; Dogs; Female; Heart Rate; Hemodynamics; Injections, Intravenous; Male; Myocardial Contraction; Ryanodine; Systole; Time Factors; Ventricular Function, Left
PubMed: 9321850
DOI: 10.1152/ajpheart.1997.273.3.H1561 -
Bioscience Reports Dec 1995The effects of ryanodine, 9,21-didehydroryanodine and 9,21-didehydroryanodol on two types of K(+) channel (a maxi, Ca(2+)-activated, 170pS channel (BK channel) and an...
The effects of ryanodine, 9,21-didehydroryanodine and 9,21-didehydroryanodol on two types of K(+) channel (a maxi, Ca(2+)-activated, 170pS channel (BK channel) and an inward rectifier, stretch sensitive channel of 35 pS conductance (IK channel)found in the plasma membrane of locust skeletal muscle have been investigated. 10(-9) M-10(-5) M ryanodine irreversibly induced a dose-dependent reduction of the reversal potential (V (rev)) of the currents of both channels, i.e. from 60 mV in the absence of the alkaloid to 15 mV for 10(-5) M ryanodine, measured under physiologically normal K(+) and Na(+) gradients. In both cases the change in the ionic selectivity was Ca(2+) -independent. 9,21-didehydroryanodine and 9,21-didehyroryanodol also reduced V (rev), but only to 35 mV during application of 10(-5) M of these compounds. Additionally, 9,21-didehydroryanodine reversibly diminished the conductances of the two K(+) channels. To test the hypothesis that ryanoids increase Na permeability by enlarging the K(+) channels, the channels were probed with quaternary ammonium ions during ryanoid application. When applied to the cytoplasmic face of inside-out patches excised from locust muscle membrane, TEA blocked the K(+) channels in a voltage-dependent fashion. The dissociation constant (K (d)(0)) for TEA block of the IK channel was reduced from 44 mM to 1 mM by 10(-7) M ryanodine, but the voltage-dependence of the block was unaffected. Qualitatively similar data were obtained for the BK channel. Ryanodine had no effect on the K (d) for cytoplasmically-applied TMA. However, the voltage-dependence for TMA block was increased for both K(+) channels, from 0.47 to 0.8 with 10(-6) M ryanodine. The effects of ryanodine on TEA and TMA block support the hypothesis that ryanodine enlarges the K(+) channels so as to facilitate permeation of partially hydrated Na(+) ions.
Topics: Animals; Bridged-Ring Compounds; Cell Membrane; Grasshoppers; In Vitro Techniques; Membrane Potentials; Muscle, Skeletal; Potassium Channel Blockers; Potassium Channels; Quaternary Ammonium Compounds; Ryanodine; Tetraethylammonium Compounds
PubMed: 9156581
DOI: 10.1007/BF01204354 -
Cell Calcium Aug 2015The earliest critical event of pancreatitis is a long lasting high amplitude rise of intracellular Ca(2+) concentration of the acinar cell, which can be triggered by...
The earliest critical event of pancreatitis is a long lasting high amplitude rise of intracellular Ca(2+) concentration of the acinar cell, which can be triggered by high concentration of bile acids. Although, Ca(2+)-release through ryanodine receptors (RyR) is involved in the process, the significance and the exact mechanism of bile acid's action on RyR has not been fully elucidated yet. Therefore, we aimed to test with various techniques and aspects whether bile acids exert a direct effect on RyR and SERCA pump. Our data show that taurocholic acid (TCA)-induced Ca(2+) release in pancreatic acinar cells was significantly reduced by the RyR antagonist dantrolene. Further, we show that TCA enhanced RyR's (3)H-ryanodine binding and triggered robust Ca(2+)-release from RyR-enriched vesicles in the pathologically relevant concentration range. RyR single channel current analysis demonstrated that 200μM TCA induced a 5-fold increase in the channel's open probability and caused a significant lengthening of the mean open time. TCA also suppressed Ca(2+)-uptake rate and ATP-ase activity of SERCA-enriched vesicles, but interestingly, failed to decrease Ca(2+) elimination rate in intact cells. Overall, our results strongly suggest that TCA opens RyR by an allosteric mechanism, which contribute significantly to bile acid-induced pathologic Ca(2+)-leak from the endoplasmic reticulum in pancreatic acinar cells.
Topics: Acinar Cells; Animals; Bile Acids and Salts; Calcium; Cholagogues and Choleretics; Dantrolene; Mice; Microsomes; Pancreas, Exocrine; Rats; Ryanodine; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Taurocholic Acid
PubMed: 25931303
DOI: 10.1016/j.ceca.2015.03.009