ryanodine and alpha-beta-methyleneadenosine-5--triphosphate

Topics: Adenosine Triphosphate; Animals; Calcium; Calcium Channel Agonists; Cyclic AMP; Humans; Liver; Models, Biological; Periodicity; Ryanodine

The detection of focal Ca(2+) transients (called neuroeffector Ca(2+) transients, or NCTs) in smooth muscle of the mouse isolated vas deferens has been used to detect the packeted release of ATP from nerve terminal varicosities acting at postjunctional P2X receptors. The present study investigates the sources and sequestration of Ca(2+) in NCTs. Smooth muscle cells in whole mouse deferens were loaded with the Ca(2+) indicator Oregon Green 488 BAPTA-1 AM and viewed with a confocal microscope. Ryanodine (10 microM) decreased the amplitude of NCTs by 45 +/- 6 %. Cyclopiazonic acid slowed the recovery of NCTs (from a time course of 200 +/- 10 ms to 800 +/- 100 ms). Caffeine (3 mM) induced spontaneous focal smooth muscle Ca(2+) transients (sparks). Neither of the T-type Ca(2+) channel blockers NiCl2 (50 microM) or mibefradil dihydrochloride (10 microM) affected the amplitude of excitatory junction potentials (2 +/- 5 % and -3 +/- 10 %) or NCTs (-20 +/- 36 % and 3 +/- 13 %). In about 20 % of cells, NCTs were associated with a local, subcellular twitch that remained in the presence of the alpha1-adrenoceptor antagonist prazosin (100 nM), showing that NCTs can initiate local contractions. Slow (5.8 +/- 0.4 microm s(-1)), spontaneous smooth muscle Ca(2+) waves were occasionally observed. Thus, Ca(2+) stores initially amplify and then sequester the Ca(2+) that enters through P2X receptors and there is no amplification by local voltage-gated Ca(2+) channels.

Topics: Adenosine Triphosphate; Animals; Caffeine; Calcium; Calcium Channels, T-Type; Calcium Signaling; Chelating Agents; Electric Stimulation; Electrophysiology; In Vitro Techniques; Indoles; Male; Mibefradil; Mice; Mice, Inbred BALB C; Microscopy, Confocal; Models, Biological; Muscle Contraction; Muscle, Smooth; Nickel; Prazosin; Purinergic P2 Receptor Antagonists; Receptors, Purinergic P2; Receptors, Purinergic P2X; Ryanodine; Thapsigargin; Vas Deferens

Bisprasin, a unique bromotyrosine derivative containing a disulfide linkage, was isolated from a marine sponge of Dysidea spp. This compound caused a concentration-dependent (from 10 to 30 microM) increase in the (45)Ca(2+) release from the heavy fraction of skeletal muscle sarcoplasmic reticulum (HSR) of rabbit skeletal muscle in the same way as does caffeine. The 50% effective concentrations of bisprasin and caffeine were approximately 18 microM and 1.2 mM, respectively, indicating that the (45)Ca(2+)-releasing activity of bisprasin was approximately 70 times more potent than that of caffeine in HSR. The bell-shaped profile of Ca(2+) dependence for bisprasin was almost the same as that for caffeine. Typical blockers of Ca(2+)-induced Ca(2+) release channels, such as Mg(2+), procaine, and ruthenium red, inhibited markedly bisprasin- and caffeine-induced (45)Ca(2+) release from HSR. This compound, like caffeine, significantly enhanced [(3)H]ryanodine binding to HSR. Scatchard analysis of [(3)H]ryanodine binding to HSR revealed that bisprasin and caffeine decreased the K(D) value without affecting the B(max) value, suggesting that both the drugs facilitate the opening of ryanodine receptor channels. The bisprasin- and caffeine-induced increases in [(3)H]ryanodine binding were further enhanced by adenosine-5'-(beta, gamma-methylene)triphosphate. These results suggest that the pharmacological properties of bisprasin are almost similar to those of caffeine, except for its 70-fold higher potency. Here, we present the first report on the pharmacological properties of bisprasin, which, like caffeine, induces Ca(2+) release from skeletal muscle SR mediated through the ryanodine receptor.

Topics: Adenosine Triphosphate; Animals; Caffeine; Calcium; Calcium Channel Blockers; Disulfides; Dose-Response Relationship, Drug; Drug Interactions; In Vitro Techniques; Magnesium; Male; Muscle, Skeletal; Porifera; Procaine; Protein Binding; Rabbits; Ruthenium Red; Ryanodine; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum; Time Factors; Tyrosine

The effects of several modulators of ryanodine receptors (RYRs) on the reduction of acetylcholine induced inward current (ACh-current) evoked by EPYLRFamide (5 microM, bath application), the potent N-terminally modified analogue of the endogenous Helix heptapeptide SEPYLRFamide, were investigated. These modulators were applied intracellularly. Inward currents were recorded from identified Helix lucorum LPa2, LPa3, RPa3, RPa2 neurones in ganglia preparations using the two-electrode voltage clamp technique. ACh was applied ionophoretically. BAPTA (0.1 mM), chelator of intracellular Ca(2+), ryanodine (0.1 mM), agonist/antagonist of RYRs and dantrolene (0.1 mM), antagonist of RYRs decrease the effect of EPYLRFamide. Adenosine (1 mM), alpha,beta-methylene ATP (0.1 mM), the nonhydrolisable ATP analogue and cyclic adenosine diphosphate ribose (0.1 mM) (agonists of RYRs) potentiate the modulatory effect of EPYLRFamide. Ruthenium red (1 mM), antagonist of RYRs and caffeine (1 mM), agonist of RYRs do not change the modulatory effect of EPYLRFamide. These data suggest that intracellular Ca(2+) and RYRs are involved in the modulatory effect of EPYLRFamide on ACh-currents. It was concluded that EPYLRFamide decreases ACh-current through elevation of basal intracellular level of a putative endogenous agonist of RYRs which activates RYR-dependent mobilization of Ca(2+) by binding to the adenine nucleotide site of the ryanodine receptor-channel complex and does not bind the site activated by caffeine.

Topics: Acetylcholine; Adenosine; Adenosine Diphosphate Ribose; Adenosine Triphosphate; Aniline Compounds; Animals; Caffeine; Chelating Agents; Cyclic ADP-Ribose; Dantrolene; Egtazic Acid; Fluorescent Dyes; Helix, Snails; Neurons; Neuropeptides; Ryanodine; Ryanodine Receptor Calcium Release Channel; Xanthenes

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.

Topics: Adenosine Triphosphate; Animals; Cell Membrane; Kinetics; Magnesium; Muscle Fibers, Skeletal; Muscle, Skeletal; Phosphates; Ryanodine; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum; Swine

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.

Topics: Adenosine Triphosphate; Animals; Antineoplastic Agents; Binding Sites; Caffeine; Calcium; Calcium Channels; Calmodulin; Cell Fractionation; Central Nervous System Stimulants; Dose-Response Relationship, Drug; Doxorubicin; Male; Muscle, Skeletal; Rabbits; Radioligand Assay; Ryanodine; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum; Subcellular Fractions; Tritium

Two distinct skeletal muscle ryanodine receptors (RyR1s) are expressed in a fiber type-specific manner in fish skeletal muscle (11). In this study, we compare [(3)H]ryanodine binding and single channel activity of RyR1-slow from fish slow-twitch skeletal muscle with RyR1-fast and RyR3 isolated from fast-twitch skeletal muscle. Scatchard plots indicate that RyR1-slow has a lower affinity for [(3)H]ryanodine when compared with RyR1-fast. In single channel recordings, RyR1-slow and RyR1-fast had similar slope conductances. However, the maximum open probability (P(o)) of RyR1-slow was threefold less than the maximum P(o) of RyR1-fast. Single channel studies also revealed the presence of two populations of RyRs in tuna fast-twitch muscle (RyR1-fast and RyR3). RyR3 had the highest P(o) of all the RyR channels and displayed less inhibition at millimolar Ca(2+). The addition of 5 mM Mg-ATP or 2.5 mM beta, gamma-methyleneadenosine 5'-triphosphate (AMP-PCP) to the channels increased the P(o) and [(3)H]ryanodine binding of both RyR1s but also caused a shift in the Ca(2+) dependency curve of RyR1-slow such that Ca(2+)-dependent inactivation was attenuated. [(3)H]ryanodine binding data also showed that Mg(2+)-dependent inhibition of RyR1-slow was reduced in the presence of AMP-PCP. These results indicate differences in the physiological properties of RyRs in fish slow- and fast-twitch skeletal muscle, which may contribute to differences in the way intracellular Ca(2+) is regulated in these muscle types.

Topics: Adenine Nucleotides; Adenosine Triphosphate; Air Sacs; Animals; Calcium; Electric Conductivity; Fishes; Muscle Fibers, Fast-Twitch; Muscle Fibers, Slow-Twitch; Muscle, Skeletal; Ryanodine; Ryanodine Receptor Calcium Release Channel; Tritium; Tuna

A rapid assay for high affinity [3H]ryanodine binding to 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS)-solubilized recombinant or native Ca2+ release channel proteins (ryanodine receptor, RyR) was devised. The key to preservation of high affinity [3H]ryanodine binding sites in the presence of increasing concentrations of CHAPS was the addition of phosphatidylcholine. This assay was used to characterize the equilibrium and kinetic properties of [3H]ryanodine binding to recombinant skeletal (RyR1) and cardiac (RyR2) Ca2+ release channels and the effects on binding of physiological modulators including ATP, Ca2+, and Mg2+. Both RyR1 and RyR2 had a single high affinity ryanodine binding site and low affinity sites, but [3H]ryanodine binding to recombinant RyR2 was not sensitive to ATP activation or Ca2+ inactivation and was less sensitive to Mg2+ inhibition. The [3H]ryanodine binding assay was used to estimate the expression level of recombinant RyR2 and RyR1, and to show that RyR2 can be expressed at very high levels in HEK-293 cells. Analysis of the properties of recombinant RyR2 and RyR1 by measurement of intracellular Fura-2 fluorescence revealed that the different properties of RyR2 and RyR1 are retained in the recombinant expressed proteins.

Topics: Adenosine Triphosphate; Animals; Caffeine; Calcium; Cell Line; DNA, Complementary; Humans; Kinetics; Magnesium; Muscle, Skeletal; Protein Binding; Rabbits; Recombinant Proteins; Ryanodine; Ryanodine Receptor Calcium Release Channel; Thapsigargin; Transfection; Tritium

Caffeine (1,3,7-trimethylxanthine) in the millimolar range is known to activate the skeletal muscle Ca2+ release channel (ryanodine receptor). Xanthine analogs substituted in the 1, 3, 7, 8 and 9 positions were tested for their capacity to increase [3H]ryanodine binding to skeletal muscle sarcoplasmic reticulum vesicles enriched in Ca2+ release activity and ryanodine receptor content. Of the 30 xanthines tested, 9 were more effective than caffeine in increasing [3H]ryanodine binding. The 7-methyl group of caffeine was most important for activating the ryanodine receptor, followed by the methyl groups in the 1 and 3 positions. An increase in hydrophobicity of the side chains in positions 7, 1 and 3 enhanced the ability of xanthines to activate the ryanodine receptor. Substitutions in positions 8 and 9 were without effect or were inhibitory. These results should help in developing xanthines specific for the sarcoplasmic reticulum Ca2+ release channel.

Topics: Adenosine Triphosphate; Animals; Binding, Competitive; Caffeine; Calcium; Calcium Channels; Calmodulin-Binding Proteins; Central Nervous System Stimulants; Dose-Response Relationship, Drug; Muscle Proteins; Muscle, Skeletal; Rabbits; Ryanodine; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum; Structure-Activity Relationship; Xanthines

Immunoblot analysis and [3H]ryanodine binding were used to characterize and identify ryanodine receptors (RyRs) in nonexcitable mouse parotid acini. Western analysis revealed ryanodine receptor type III (Ry3R) to be the only detectable isoform in parotid microsomal membranes. Binding of [3H]ryanodine to microsomal fractions was dependent on Ca2+, salt, pH, and temperature. At 23 degrees C, and in the presence of 0.5 M KCl and 100 microM Ca2+, [3H]ryanodine bound specifically to membranes with high affinity (Kd = 6 nM); maximum binding capacity (Bmax) was 275 fmol/mg protein. Mg2+ and ruthenium red inhibited [3H]ryanodine binding (IC50 = 1.4 mM and 0.5 microM, respectively). 4-Chloro-3-ethylphenol enhanced the binding of [3H]ryanodine 2.5-fold; whereas ATP and caffeine were much less efficacious toward activating Ry3R (56% and 18% maximal enhancement, respectively). Bastadin, a novel modulator of the 12-kDa FK506 binding protein.RyR complex, increased [3H]ryanodine binding 3-4-fold by enhancing Kd. The immunosuppressant FK506 enhanced [3H]ryanodine receptor occupancy at >100 microM and antagonized the action of bastadin, suggesting that an immunophilin modulates Ry3R in parotid acini. These results suggest that Ry3R may play an important role in Ca2+ homeostasis in mouse parotid acini.

Topics: Adenosine Triphosphate; Animals; Binding Sites; Blotting, Western; Caffeine; Calcium Channels; Calmodulin-Binding Proteins; Kinetics; Magnesium Chloride; Mice; Microsomes; Muscle Proteins; Parotid Gland; Ryanodine; Ryanodine Receptor Calcium Release Channel

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.

Topics: Adenosine Triphosphate; Animals; Caffeine; Calcium; Calcium Channels; Cholic Acids; Muscle Proteins; Muscle, Skeletal; Phospholipids; Rana catesbeiana; Ryanodine; Ryanodine Receptor Calcium Release Channel; Sodium Chloride; Tritium

[3H]Ryanodine binding to, as well as functions of, ryanodine receptor intracellular Ca2+ release channel complexes are modulated by several adenosine-based compounds. In this study, we determined the effects of endogenous compounds termed diadenosine polyphosphates (ApnAs; n = 2-6 phosphate groups) on [3Hlryanodine binding to membranes prepared from rat brain and skeletal and cardiac muscle. Under low ionic strength buffer conditions, [3H]ryanodine binding to brain membranes was significantly increased by 171% with 333 microM P1,P5-di(adenosine-5') pentaphosphate (Ap5A) and by 209% with the same concentration of the metabolism-resistant ATP analogue betagamma-methyleneadenosine 5'-triphosphate (AMP-PCP) compared with control values for [3H]ryanodine binding of 9.6 +/- 1.8 fmol/mg of protein. Dose-related increases in [3H]ryanodine binding were observed for all five ApnAs tested [P1,P2-di(adenosine-5') pyrophosphate (Ap2A), P1,P3-di(adenosine-5') triphosphate (Ap3A), P1,P4-di(adenosine-5') tetraphosphate (Ap4A), Ap5A, and P1,P6-di(adenosine-5') hexaphosphate (Ap6A)] as well as AMP-PCP; oxidized salts of ApnAs stimulated [3H]ryanodine binding to a greater degree than did nonoxidized APnAs. The apparent rank order for the capacity of these agents to increase [3H]-ryanodine binding was oxidized Ap4A = oxidized Ap5A > oxidized Ap3A > Ap6A > AMP-PCP > Ap5A > AP2A. Addition of the approximate EC50 dose of oxidized Ap4A (37 microM) increased the affinity (KD) of ryanodine receptors from 34 +/- 7 to 12 +/- 2 nM, the apparent binding site density (Bmax) was not significantly different from control values of 107 +/- 33 fmol/mg of protein. Increases in [3H]-ryanodine binding by either oxidized Ap4A or nonoxidized Ap5A were not further enhanced by coincubation with AMP-PCP, which suggests a similar site of action for the ApnAs and AMP-PCP. [3H]Ryanodine binding to skeletal and cardiac muscle membranes was enhanced by addition of oxidized AP4A Ap5A, and AMP-PCP. Oxidized AP4A increased the specific binding by ninefold in skeletal muscle and by threefold in cardiac muscle. These results suggest that ApnAs, at physiologically relevant concentrations, may serve as endogenous modulators of ryanodine receptor-gated Ca2+ release channels.

Topics: Adenosine Triphosphate; Animals; Calcium; Calcium Channels; Cerebral Cortex; Dinucleoside Phosphates; Ion Channel Gating; Kinetics; Male; Muscle Proteins; Muscle, Skeletal; Myocardium; Oxidation-Reduction; Rats; Rats, Sprague-Dawley; Ryanodine; Ryanodine Receptor Calcium Release Channel; Structure-Activity Relationship

[3H]9-Methyl-7-bromoeudistomin D ([3H]MBED), the most powerful Ca2+ releaser from sarcoplasmic reticulum, specifically bound to the brain microsomes. Caffeine competitively inhibited [3H]MBED binding. [3H]MBED binding was markedly blocked by procaine, whereas that was enhanced by adenosine-5'-(beta,gamma-methylene)triphosphate. The Bmax value was 170 times more than that of [3H]ryanodine binding. The profile of sucrose-density gradient centrifugation of solubilized microsomes indicated that [3H]MBED binding protein was different from [3H]ryanodine binding protein. These results suggest that there are MBED/caffeine-binding sites in brain that are distinct from the ryanodine receptor and that MBED becomes an essential molecular probe for characterizing caffeine-binding protein in the central nervous system.

Topics: Adenosine Triphosphate; Animals; Binding Sites; Binding, Competitive; Brain; Caffeine; Calcium; Calcium Channels; Carbolines; Cholic Acids; Cyclic GMP; Detergents; Guinea Pigs; Inositol 1,4,5-Trisphosphate; Kinetics; Microsomes; Muscle Proteins; Procaine; Ruthenium Red; Ryanodine; Ryanodine Receptor Calcium Release Channel

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.

Topics: Adenosine Triphosphate; Animals; Ca(2+) Mg(2+)-ATPase; Caffeine; Calcium; Calcium Radioisotopes; Crotalid Venoms; In Vitro Techniques; Ion-Selective Electrodes; Muscle, Skeletal; Procaine; Rabbits; Ryanodine; Sarcoplasmic Reticulum

Suramin is a polysulfonated naphthylurea developed originally to treat trypanosomiasis. This drug has gained considerable attention recently as an effective anticancer agent. Previous studies have demonstrated that suramin also is an antagonist of ATP at P2x purinergic receptors. In the present study suramin was shown to evoke Ca++ release from skeletal muscle sarcoplasmic reticulum (SR) vesicles in a concentration-dependent manner. Ca++ release was inducable from vesicles derived from junctional SR but not from those derived from longitudinal SR. This subcellular site-dependent specificity suggests that suramin's actions on muscle involve the Ca++ release channel (CRC), a protein unique to terminal cisternae. This channel has been established as the site of action of ryanoid alkaloids such as ryanodine and dehydroryanodine. Suramin did not mimic ryanoid actions on the SR CRC, nor did it competitively diminish ryanodine binding. Instead, suramin actually increased [3H]ryanodine binding to junctional SR membranes. In this respect, suramin exhibited agonist effects like those of the adenine nucleotide, beta,gamma-methyleneadenosine 5'-triphosphate. Suramin's mechanism of action did not involve oxidation of sulfhydryl groups on the SR CRC, because dithiothreitol (1 mM) had no effect on suramin-induced Ca++ release. Independently of its effects on the CRC, suramin inhibited the Ca(++)-adenosine triphosphatase (EC 3.6.1.38, SERCA1) of SR membrane vesicles. The ability of suramin to diminish ATP-dependent Ca++ accumulation by SR vesicles therefore reflects two distinct actions: 1) activation (opening) of the SR Ca++ release channel and 2) inhibition of the Ca++ pump.

Topics: Adenosine Triphosphate; Animals; Biological Transport; Calcium; Calcium Channels; Calcium-Transporting ATPases; Dithiothreitol; Drug Interactions; In Vitro Techniques; Rabbits; Ryanodine; Sarcoplasmic Reticulum; Suramin

Micromolar concentrations of the porphyrin mesotetra(4-N-methylpyridyl)porphine tetraiodide is shown to induce rapid release of Ca2+ from skeletal muscle sarcoplasmic reticulum vesicles. Porphyrin-induced Ca2+ release is stimulated by ATP (KdATP = 100 microM) and Ca2+ (KdCa = 1 microM) and is inhibited by Mg2+ (KI = 220 microM) and ruthenium red (KI = 7 nM). The porphyrin is also shown to stimulate high affinity [3H]ryanodine binding by decreasing the dissociation constant (kd) and increasing the binding capacity (Bmax). Moreover, in the presence of Mg2+, receptor binding is sensitized to activation by Ca2+, and porphyrin-stimulated channel activity is sensitized to activation by Ca2+. These observations show that porphyrin-induced Ca2+ release is due to a direct interaction with the Ca2+ release protein from sarcoplasmic reticulum.

Topics: Adenosine Triphosphate; Animals; Caffeine; Calcium; Calcium Channels; Dithiothreitol; Magnesium; Mesoporphyrins; Porphyrins; Rabbits; Ruthenium Red; Ryanodine; Sarcoplasmic Reticulum

3H-Labeled 9-methyl-7-bromoeudistomin D ([3H] MBED), the most powerful inducer of Ca2+ release from sarcoplasmic reticulum (SR), was successfully prepared with a high specific activity of 10.2 Ci/mmol. [3H]MBED bound to terminal cisternae (TC) of skeletal muscle SR in a replacable and saturable manner, indicating the existence of its specific binding site. Caffeine inhibited the [3H]MBED binding to the TC-SR membranes from skeletal muscle with an IC50 value of 0.8 mM, in close agreement with a concentration that causes Ca2+ release from SR. Scatchard analysis gave values of KD = 40 nM and Bmax = 10 pmol/mg protein. The KD value was increased by caffeine, while that of Bmax was not changed, indicating a competitive mode of inhibition. Adenosine 5'-(beta, gamma-methylene)triphosphate enhanced [3H]MBED binding, but ryanodine and Ca2+ did not affect it. [3H]MBED binding to TC-SR membranes was inhibited by procaine, a representative blocker of Ca(2+)-induced Ca2+ release channels, whereas that was not changed by Mg2+, suggesting that procaine but not Mg2+ may exert its inhibitory effect on Ca(2+)-induced Ca2+ release by affecting the caffeine-binding sites. These results suggest that MBED shares the same binding site as that of caffeine in TC-SR. The [3H]MBED is the first radiolabeled ligand for caffeine-binding sites in Ca2+ release channels and thus may provide an essential biochemical tool for elucidating this site.

Topics: Adenosine Triphosphate; Animals; Binding Sites; Binding, Competitive; Caffeine; Calcium; Carbolines; Cell Membrane; Magnesium; Muscles; Procaine; Rabbits; Ruthenium Red; Ryanodine; Sarcoplasmic Reticulum

It has been clarified that ryanodine binds to Ca2(+)-induced Ca release channels in the open state in sarcoplasmic reticulum. While the pharmacological action of ryanodine is known to be retarded at a low temperature, the Ca-releasing action of caffeine is potentiated at a low temperature. In order to obtain deeper insight into the molecular mechanism underlying Ca-release, the effect of temperature on ryanodine binding to the heavy fraction of sarcoplasmic reticulum (HFSR) from bullfrog skeletal muscle was examined. Although Ca2+ is indispensable for ryanodine binding, Ca2+ alone cannot cause ryanodine binding in a reaction medium of a salt concentration similar to that of the sarcoplasm. In addition to Ca2+, caffeine and/or beta,gamma-methylene adenosine triphosphate (AMPOPCP) are necessary. [3H]Ryanodine binding at 25 degrees C closely paralleled the Ca release activity in respect of the Ca2(+)-dependence in the presence of caffeine and/or AMPOPCP, and the effects of inhibitors. A Scatchard plot for ryanodine binding gave a straight linear line, indicating a single class of homogeneous binding sites. At 0 degrees C, the rate of ryanodine binding decreased. Q10 being about 3 on average. The affinity for ryanodine was reduced to about half that at 25 degrees C, with no change in the maximum number of binding sites. The temperature-dependent change in apparent affinity for Ca2+ on ryanodine binding is not always consistent with that in the case of Ca-release activity. The bound ryanodine may be in an occluded state because it did not dissociate for up to 90 h at 0 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenosine Triphosphate; Alkaloids; Animals; Caffeine; Calcium; Rana catesbeiana; Ryanodine; Sarcoplasmic Reticulum; Temperature

While many reports have shown that Ca2+ alone causes ryanodine binding to the heavy fraction of the sarcoplasmic reticulum (HFSR), our results demonstrate that caffeine or beta,gamma-methylene adenosine triphosphate (AMPOPCP) in addition to Ca2+ is necessary for ryanodine binding, although Ca2+ is indispensable for it. While clarifying the reasons for this discrepancy, we found that a high osmolarity of the reaction medium, but not ionic strength, is a crucial factor. In a hypertonic solution containing 1 M NaCl, Ca2+ alone causes a sizable extent of ryanodine binding. Caffeine and AMPOPCP independently stimulate it, unlike the case of 0.17 M KCl (or NaCl) medium, in which they show a potentiating interaction. Ryanodine binding in the hypertonic solution was markedly enhanced not only as to the binding rate but also the extent. The Scatchard plot was linear, indicating a single class of homogeneous binding sites. The maximum number of binding sites as well as the affinity was also increased in 1 M NaCl-medium. The presence of AMPOPCP and/or caffeine did not affect the magnitudes of them so much, especially that of the affinity, in the hypertonic medium, as in the isotonic medium. The Ca2(+)-dependence of ryanodine binding in the stimulatory range was similar to that in 0.17 M KCl- (or NaCl-) medium. However, the very weak inhibition at high Ca2+ concentrations is in striking contrast to ryanodine binding in the isotonic medium. The stimulation due to a high osmolarity is distinct, as to the mechanism, from that due to AMPOPCP, caffeine, or temperature. The dissociation of [3H]ryanodine bound was also examined under various experimental conditions.

Topics: Adenosine Triphosphate; Alkaloids; Animals; Caffeine; Hypertonic Solutions; Osmolar Concentration; Rana catesbeiana; Ryanodine; Sarcoplasmic Reticulum

The anthraquinones, doxorubicin, mitoxantrone, daunorubicin and rubidazone are shown to be potent stimulators of Ca2+ release from skeletal muscle sarcoplasmic reticulum (SR) vesicles and to trigger transient contractions in chemically skinned psoas muscle fibers. These effects of anthraquinones are the direct consequence of their specific interaction with the [3H] ryanodine receptor complex, which constitutes the Ca2+ release channel from the triadic junction. In the presence of adenine nucleotides and physiological Mg2+ concentrations (approximately 1.0 mM), channel activation by doxorubicin and daunorubicin exhibits a sharp dependence on submicromolar Ca2+ which is accompanied by a selective, dose-dependent increase in the apparent affinity of the ryanodine binding sites for Ca2+, in a manner similar to that previously reported with caffeine. Unlike caffeine, however, anthraquinones increase [3H]ryanodine receptor occupancy to the level observed in the presence of adenine nucleotides. A strong interaction between the anthraquinone and the caffeine binding sites on the Ca2+ release channel is also observed when monitoring Ca2+ fluxes across the SR. Millimolar caffeine both inhibits anthraquinone-stimulated Ca2+ release, and reduces anthraquinone-stimulated [3H]ryanodine receptor occupancy, without changing the effective binding constant of the anthraquinone for its binding site. The degree of cooperativity for daunorubicin activation of Ca2+ release from SR also increases in the presence of caffeine. These results demonstrate that the mechanism by which anthraquinones stimulate Ca2+ release is caused by a direct interaction with the [3H]ryanodine receptor complex, and by sensitization of the Ca2+ activator site for Ca2+.

Topics: Adenosine Triphosphate; Animals; Anthraquinones; Binding Sites; Caffeine; Calcium; Daunorubicin; Dose-Response Relationship, Drug; Doxorubicin; Magnesium; Mathematics; Mitoxantrone; Muscle Contraction; Muscles; Rabbits; Ryanodine; Sarcoplasmic Reticulum