High Concentration Of Ryanodine Research Articles

Ca(2+) release from ryanodine-sensitive store contributes to mechanism of hypoxic vasoconstriction in rat lungs.

Studies of thapsigargin, cyclopiazonic acid, and ryanodine in isolated pulmonary arteries and smooth muscle cells suggest that release of Ca(2+) from inositol 1,4,5-trisphosphate (IP(3))- and/or ryanodine-sensitive sarcoplasmic reticulum Ca(2+) stores is a component of the mechanism of acute hypoxic pulmonary vasoconstriction (HPV). However, the actions of these agents on HPV in perfused lungs have not been reported. Thus we tested effects of thapsigargin and cyclopiazonic acid, inhibitors of sarcoplasmic reticulum Ca(2+)-ATPase, and of ryanodine, an agent that either locks the ryanodine receptor open or blocks it, on HPV in salt solution-perfused rat lungs. After inhibition of cyclooxygenase and nitric oxide synthase, thapsigargin (10 nM) and cyclopiazonic acid (5 microM) augmented the vasoconstriction to 0% but not to 3% inspired O(2). Relatively high concentrations of ryanodine (100 and 300 microM) blunted HPV in nitric oxide synthase-inhibited lungs. The results indicate that release of Ca(2+) from the ryanodine-sensitive, but not the IP(3)-sensitive, store, contributes to the mechanism of HPV in perfused rat lungs and that Ca(2+)-ATPase-dependent Ca(2+) buffering moderates the response to severe hypoxia.

Effects of ryanodine on calcium sparks in cut twitch fibres of Rana temporaria.

1. Localized calcium release events (calcium sparks) were studied in voltage-clamped cut twitch fibres of Rana temporaria. 2. A histogram of thousands of spontaneous sparks displayed a monotonically decreasing amplitude distribution from the low to the high limit of > 7 DeltaF/F(0) units. 3. Several effects of low micromolar concentrations of ryanodine (0.4-2 microM) on spontaneous sparks, reproducing the agent's effects on single ryanodine receptor channel current in bilayers, were observed collectively for the first time in live fibres, namely (a) increases in spark frequency followed by (b) conversions of sparks into steady glows lasting tens of seconds, (c) occasional interruptions of the glows by brief gaps of darkness, and (d) abolition of sparks at the locations of the glows. The glow could reflect the incessant Ca(2+) flux through a single (or a few) calcium release channel locked in the semi-open state, which was allowed to make occasional transitions to the closed state but not to the fully open state. 4. Higher concentrations of ryanodine (> or = 20 microM) suppressed the spontaneous sparks effectively and permanently, presumably by deactivating the ryanodine receptors. 5. Depolarization-evoked sparks elicited with small pulses had higher frequencies and larger amplitudes than spontaneous sparks and were abolished by both concentrations of ryanodine. 6. With 1-2 microM ryanodine, however, a uniform non-sparking calcium release persisted during the pulse, with the globally averaged increase in fluorescence intensity being about half that of the control. A possible origin of this non-sparking release may be related to the structural coupling between the voltage sensors and the ryanodine receptors that can exist only in live fibres but not in the bilayer preparation.

CADP-ribose activates reconstituted ryanodine receptors from coronary arterial smooth muscle.

The present study was designed to test the hypothesis that cADP-ribose (cADPR) increases Ca(2+) release through activation of ryanodine receptors (RYR) on the sarcoplasmic reticulum (SR) in coronary arterial smooth muscle cells (CASMCs). We reconstituted RYR from the SR of CASMCs into planar lipid bilayers and examined the effect of cADPR on the activity of these Ca(2+) release channels. In a symmetrical cesium methanesulfonate configuration, a 245 pS Cs(+) current was recorded. This current was characterized by the formation of a subconductance and increase in the open probability (NP(o)) of the channels in the presence of ryanodine (0.01-1 microM) and imperatoxin A (100 nM). A high concentration of ryanodine (50 microM) and ruthenium red (40-80 microM) substantially inhibited the activity of RYR/Ca(2+) release channels. Caffeine (0.5-5 mM) markedly increased the NP(o) of these Ca(2+) release channels of the SR, but D-myo-inositol 1,4,5-trisphospate and heparin were without effect. Cyclic ADPR significantly increased the NP(o) of these Ca(2+) release channels of SR in a concentration-dependent manner. Addition of cADPR (0.01 microM) into the cis bath solution produced a 2.9-fold increase in the NP(o) of these RYR/Ca(2+) release channels. An eightfold increase in the NP(o) of the RYR/Ca(2+) release channels (0.0056 +/- 0.001 vs. 0.048 +/- 0.017) was observed at a concentration of cADPR of 1 microM. The effect of cADPR was completely abolished by ryanodine (50 microM). In the presence of cADPR, Ca(2+)-induced activation of these channels was markedly enhanced. These results provide evidence that cADPR activates RYR/Ca(2+) release channels on the SR of CASMCs. It is concluded that cADPR stimulates Ca(2+) release through the activation of RYRs on the SR of these smooth mucle cells.

Induction of neuronal apoptosis by thiol oxidation: putative role of intracellular zinc release.

The membrane-permeant oxidizing agent 2,2'-dithiodipyridine (DTDP) can induce Zn(2+) release from metalloproteins in cell-free systems. Here, we report that brief exposure to DTDP triggers apoptotic cell death in cultured neurons, detected by the presence of both DNA laddering and asymmetric chromatin formation. Neuronal death was blocked by increased extracellular potassium levels, by tetraethylammonium, and by the broad-spectrum cysteine protease inhibitor butoxy-carbonyl-aspartate-fluoromethylketone. N,N,N', N'-Tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN) and other cell-permeant metal chelators also effectively blocked DTDP-induced toxicity in neurons. Cell death, however, was not abolished by the NMDA receptor blocker MK-801, by the intracellular calcium release antagonist dantrolene, or by high concentrations of ryanodine. DTDP generated increases in fluorescence signals in cultured neurons loaded with the zinc-selective dye Newport Green. The fluorescence signals following DTDP treatment also increased in fura-2- and magfura-2-loaded neurons. These responses were completely reversed by TPEN, consistent with a DTDP-mediated increase in intracellular free Zn(2+) concentrations. Our studies suggest that under conditions of oxidative stress, Zn(2+) released from intracellular stores may contribute to the initiation of neuronal apoptosis.

A Fundamental Role for the Nitric Oxide-G-Kinase Signaling Pathway in Mediating Intercellular Ca2+Waves in Glia

In this study, we highlight a role for the nitric oxide-cGMP-dependent protein kinase (NO-G-kinase) signaling pathway in glial intercellular Ca(2+) wave initiation and propagation. Addition of the NO donor molsidomine (100-500 microM) or puffing aqueous NO onto primary glial cell cultures evoked an increase in [Ca(2+)](i) in individual cells and also local intercellular Ca(2+) waves, which persisted after removal of extracellular Ca(2+). High concentrations of ryanodine (100-200 microM) and antagonists of the NO-G-kinase signaling pathway essentially abrogated the NO-induced increase in [Ca(2+)](i), indicating that NO mobilizes Ca(2+) from a ryanodine receptor-linked store, via the NO-G-kinase signaling pathway. Addition of 10 microM nicardipine to cells resulted in a slowing of the molsidomine-induced rise in [Ca(2+)](i), and inhibition of Mn(2+) quench of cytosolic fura-2 fluorescence mediated by a bolus application of 2 microM aqueous NO to cells, indicating that NO also induces Ca(2+) influx in glia. Mechanical stress of individual glial cells resulted in an increase in intracellular NO in target and neighboring cells and intercellular Ca(2+) waves, which were NO, cGMP, and G-kinase dependent, because incubating cells with nitric oxide synthase, guanylate cyclase, and G-kinase inhibitors, or NO scavengers, reduced Delta[Ca(2+)](i) and the rate of Ca(2+) wave propagation in these cultures. Results from this study suggest that NO-G-kinase signaling is coupled to Ca(2+) mobilization and influx in glial cells and that this pathway plays a fundamental role in the generation and propagation of intercellular Ca(2+) waves in glia.

Phenylarsine Oxide Evokes Intracellular Calcium Increases and Amylase Secretion in Isolated Rat Pancreatic Acinar Cells

The effects of the thiol reagent, phenylarsine oxide (PAO, 10 −5−10 −3 M ), a membrane-permeable trivalent arsenical compound that specifically complexes vicinal sulfhydryl groups of proteins to form stable ring structures, were studied by monitoring intracellular free calcium concentration ([Ca 2+] i) and amylase secretion in collagenase dispersed rat pancreatic acinar cells. PAO increased [Ca 2+] i by mobilizing calcium from intracellular stores, since this increase was observed in the absence of extracellular calcium. PAO also prevented the CCK-8-induced signal of [Ca 2+] i and inhibited the oscillatory pattern initiated by aluminium fluoride (AlF − 4). In addition to the effects of PAO on calcium mobilization, it caused a significant increase in amylase secretion and reduced the secretory response to either CCK-8 or AlF − 4. The effects of PAO on both [Ca 2+] i and amylase release were reversed by the sulfhydryl reducing agent, dithiothreitol (2 mM). Pretreatment of acinar cells with high concentration of ryanodine (50 μM) reduced the PAO-evoked calcium release. However, PAO was still able to release a small fraction of Ca 2+ from acinar cells in which agonist-releasable Ca 2+ pools had been previously depleted by thapsigargin (0.5 μM) and ryanodine receptors were blocked by 50 μM ryanodine. We conclude that, in pancreatic acinar cells, PAO mainly releases Ca 2+ from the ryanodine-sensitive calcium pool and consequently induces amylase secretion. These effects are likely to be due to the oxidizing effects of this compound.

Changes in ryanodine receptor-mediated calcium release during skeletal muscle differentiation.

We have observed a disparity between the actions of caffeine and ryanodine, two agents known to affect the same site of intracellular calcium (Ca2+) release in muscle. The site of intracellular Ca2+ release, the ryanodine receptor (RyR), is established as the route of Ca2+ movement from the sarcoplasmic reticulum (SR) to the cytosol during excitation-contraction coupling. We measured Ca2+ release fluorimetrically in both saponin-permeabilized and intact L6 cells, in response to known modulators (i.e., caffeine and ryanodine), during differentiation in vitro. The undifferentiated L6 cells showed little response to caffeine. However, a substantial caffeine-induced calcium release (caffCR) was evident by Day 3 of differentiation, and was nearly maximal by Day 7 of differentiation. By contrast, ryanodine failed to stimulate Ca2+ release until Day 4, lagging behind the caffeine response. Ryanodine-stimulated Ca2+ release was also maximal by Day 7. Higher concentrations of ryanodine, known to inhibit Ca2+ release, only began to affect caffCR at Day 4, indicating that cells were insensitive to both ryanodine stimulation and ryanodine inhibition prior to this time. Most of the results could be obtained both in permeabilized and intact cells. Using intact cells, we measured the time course of K+ -dependent (i.e., depolarization-induced) Ca2+ release. This time course matched caffeine and not ryanodine-induced Ca2+ release suggesting the action of caffeine was not due to Ca2+ release unrelated to excitation-contraction coupling. These findings suggest that ryanodine binding sites on the RyR may not be functional at early stages of muscle development, that ryanodine sensitivity is a poor indicator of Ca2+ flux through the RyR, or that other proteins are involved in Ca2+ release under certain circumstances.

Changes in Ryanodine Receptor-Mediated Calcium Release During Skeletal Muscle Differentiation

We have observed a disparity between the actions of caffeine and ryanodine, two agents known to affect the same site of intracellular calcium (Ca2+) release in muscle. The site of intracellular Ca2+ release, the ryanodine receptor (RyR), is established as the route of Ca2+ movement from the sarcoplasmic reticulum (SR) to the cytosol during excitation-contraction coupling. We measured Ca2+ release fluorimetri-cally in both saponin-permeabilized and intact L6 cells, in response to known modulators (i.e., caffeine and ryanodine), during differentiation in vitro. The undifferentiated L6 cells showed little response to caffeine. However, a substantial caffeine-induced calcium release (caffCR) was evident by Day 3 of differentiation, and was nearly maximal by Day 7 of differentiation. By contrast, ryanodine failed to stimulate Ca2+ release until Day 4, lagging behind the caffeine response. Ryanodine-stimulated Ca2+ release was also maximal by Day 7. Higher concentrations of ryanodine, known to inhibit Ca2+ release, only began to affect caffCR at Day 4, indicating that cells were insensitive to both ryanodine stimulation and ryanodine inhibition prior to this time. Most of the results could be obtained both in permeabilized and intact cells. Using intact cells, we measured the time course of K+-dependent (i.e., depolarization-induced) Ca2+ release. This time course matched caffeine and not ryanodine-induced Ca2+ release suggesting the action of caffeine was not due to Ca2+ release unrelated to excitation-contraction coupling. These findings suggest that ryanodine binding sites on the RyR may not be functional at early stages of muscle development, that ryanodine sensitivity is a poor indicator of Ca2+ flux through the RyR, or that other proteins are involved in Ca2+ release under certain circumstances.

NADH-Linked Substrate-Mediated Enhancement of Mitochondrial Calcium Accumulation and DNA Single-Strand Breakage Elicited bytert-Butylhydroperoxide: The Source of the Cation Is a Ryanodine-Sensitive Calcium Store

We previously found that the membrane-permeant NADH-linked substrates pyruvate and β-hydroxybutyrate enhance the formation of DNA single-strand breaks induced bytert-butylhydroperoxide (tB-OOH) in intact U937 cells. This effect is mediated by a process involving enforced mitochondrial calcium accumulation in the absence of discernible elevation in the cytosolic concentration of free calcium ions. We now show that the intracellular source of the cation is a ryanodine-sensitive calcium store. A high concentration of ryanodine, which suppressed the caffeine-mediated mobilization of calcium ions, also abolished the effects of the NADH-linked substrates on the mitochondrial accumulation of the cation as well as on the tB-OOH-induced genotoxic response. These data constitute a novel demonstration of a physiological mechanism with important pathological implications.

The Cardiac Content of Sarcoplasmic Reticulum in the Rat Determined by Calcium Uptake Rate, Calcium Oxalate Capacity, Ryanodine Binding and Thapsigargin Titration

The amount of cardiac sarcoplasmic reticulum in rat hearts was estimated by comparing marker activities in the isolated SR fraction with their activities in the homogenate. Four distinguishable markers were measured: the oxalate-supported rate of calcium uptake, the calcium oxalate capacity,3H-ryanodine binding and the thapsigargin equivalents. The calcium uptake rate and capacity and thapsigargin equivalents were determined in the presence and absence of SR Ca2+channel blockade with high concentrations of ryanodine. All of these activities are believed to be located only in the SR. However, the calculation of the heart content of SR was somewhat different for the four markers. The calcium uptake rate gave 8.4 mg SR protein per g tissue in the absence of ryanodine, and 9.6 mg per g in its presence; calcium oxalate capacity gave similar numbers, 9.9 mg per g in the absence of ryanodine and 8.0 mg per g in its presence. The thapsigargin titration gave similar equivalent with or without ryanodine, indicating that the homogenate contained about 8.0 mg of SR per g tissue. Using3H-ranodine binding as a marker, the cardiac content of SR was calculated to be 16.7 mg per g. These differences are attributed to the non-ideal behavior of these markers. Some of the Ca2+uptake activity is not thapsigargin sensitive, and some of the3H-ryanodine binding does not fractionate with the SR Ca2+uptake activity.

Calcium channel subtypes responsible for voltage-gated intracellular calcium elevations in embryonic rat motoneurons

The central role of electrical activity and Ca 2+ influx in motoneuron development raises important questions about the regulation of Ca 2+ signalling induced by voltage-dependent Ca 2+ influx. In the purified embryonic rat motoneuron preparation, we recorded barium currents through voltage-activated Ca 2+ channels using the whole-cell configuration of the patch-clamp technique. We found that motoneurons express at least four types of high-voltage-activated Ca 2+ channels, based on their kinetics, voltage-dependences and pharmacological properties. Of the sustained Ca 2+ current activated at 0 mV from a holding potential of −100 mV, approximatively 45% was omega-conotoxin-GVIA (1 μM) sensitive, 25% was omega-agatoxin-IVA (30 nM) sensitive and 20% was nitrendipine (250 nM) sensitive. The residual current, after applying these three antagonists, was an inactivating current that differs from classical T-type Ca 2+ currents. Based on this pharmacology, changes in intracellular free Ca 2+ concentrations were then monitored by Fura 2 digital imaging microspectrofluorimetry. Upon K + depolarization, the intracellular Ca 2+ transient induced by the activation of each type of Ca 2+ channel appeared to be quantitatively proportional to their Ca 2+ influx. The existence of a calcium-induced calcium release mechanism through activation of caffeine-, ryanodine-sensitive intracellular stores was then investigated. High doses of caffeine and low doses of ryanodine failed to increase intracellular free calcium concentrations and low concentrations of caffeine and high concentrations of ryanodine did not affect K +-induced intracellular free calcium concentration transients indicating both the absence of Ca 2+-gated Ca 2+-release channels and of a Ca 2+-induced Ca 2+ release mechanism. Together, these data provide evidence that embryonic motoneurons express multiple Ca 2+ channels that function as important regulators of intracellular Ca 2+ signalling and may be involved in their development.

Ryanodine-sensitive, thapsigargin-insensitive calcium uptake in rat ventricle homogenates.

Thapsigargin is a natural product that specifically inhibits all known SERCA calcium pumps with high affinity. We investigated the effects of thapsigargin on cardiac sarcoplasmic reticulum (SR) by measuring the oxalate-supported calcium uptake rate in the unfractionated homogenate and in the isolated SR fraction. The uptake rate in both the isolated SR and unfractionated homogenate are stimulated about two-fold by preincubation with high concentrations of ryanodine, which closes the SR efflux channel. Thapsigargin stoichiometrically and completely inhibited the calcium uptake rate in the isolated SR, both in the presence and absence of SR channel blockade. In contrast, thapsigargin nearly completely inhibited the homogenate calcium uptake only in the absence of SR channel blockade; in the presence of blockade, about 20% of the uptake activity was insensitive to thapsigargin. This result unmasks a thapsigargin-insensitive, ryanodine-sensitive component of calcium uptake in the heart. This activity is in an oxalate-permeable pool and is inhibited by cyclopiazonic acid, another inhibitor of the SERCA calcium pumps. There was no TG-insensitive activity in the rat EDL muscle homogenate. The absence of thapsigargin-insensitive uptake activity in the isolated SR can be attributed to its inactivation during the isolation of the SR. The oxalate permeability and ryanodine sensitivity suggest that the TG-insensitive calcium uptake activity is closely related to the classical SR. The different thapsigargin sensitivities suggests the existence of two kinds of intracellular calcium pumps in the heart.

Characterization of ryanodine-sensitive Ca2+ release from microsomal vesicles of rat parotid acinar cells: regulation by cyclic ADP-ribose.

We have measured ryanodine (caffeine)-sensitive 45Ca2+ release from isolated microsomal vesicles of endoplasmic reticulum prepared from rat parotid acinar cells. After a steady state of ATP-dependent 45Ca2+ uptake, the addition of caffeine (40 mm), ryanodine (10 approximately 500 microm) or an NAD+ metabolite, cyclic ADP-ribose (cADPR, 4 microm) released about 10% of the 45Ca2+ that had been taken up. The 45Ca2+ release was not inhibited by heparin, an antagonist of IP3 receptor. The effects of caffeine, ryanodine and cADPR on 45Ca2+ release were also tested in the presence of thapsigargin (TG), an inhibitor of microsomal Ca2+-ATPase. When caffeine (10 approximately 40 mm), ryanodine (10 microm) or cADPR (1 approximately 10 microm) was added in the medium with 100 nm TG, a significant 45Ca2+ release was seen, while higher concentrations of ryanodine (>100 microm) did not cause any 45Ca2+ release in the presence of TG. The initial rate of caffeine (40 mm)-induced 45Ca2+ release was increased by a pretreatment with 10 microm ryanodine, whereas the caffeine-induced 45Ca2+ release was strongly inhibited by the presence of a higher concentration (500 microm) of ryanodine. cADPR-induced 45Ca2+ release was also inhibited by 500 microm ryanodine. Caffeine (40 mm)- or cADPR (4 microm)-induced 45Ca2+ release was abolished by a presence of ruthenium red (50 approximately 100 microm). The presence of a low concentration (0.5 microm) of cADPR shifted the dose-response curve of caffeine-induced 45Ca2+ release to the left. These results indicate the presence of a ryanodine sensitive Ca2+ release mechanism in the endoplasmic reticulum of rat parotid acinar cells that is distinct from the IP3-sensitive Ca2+ channel and is activated by caffeine, cADPR and a low concentration (10 microm) of ryanodine, but is inhibited by higher concentrations (>100 microm) of ryanodine and ruthenium red. The properties of the ryanodine-sensitive mechanism are similar to that of the ryanodine receptor as described in muscle cells.

Thiol oxidation by 2,2'-dithiodipyridine causes a reversible increase in cytoplasmic free Ca2+ concentration in pancreatic beta-cells. Role for inositol 1,4,5-trisphosphate-sensitive Ca2+ stores.

2,2'-Dithiodipyridine (2,2'-DTDP), a reactive disulphide that mobilizes Ca2+ from ryanodine-sensitive Ca2+ stores in muscle, induced a biphasic increase in cytoplasmic free Ca2+ concentration ([Ca2+]i) in pancreatic beta-cells loaded with fura 2. This increase consisted of an early transient followed by a second, slower, rise. The [Ca2+]i transient was dependent on extracellular Ca2+ and disappeared on treatment with nimodipine. The reactive disulphide caused plasma membrane depolarization, as studied by the perforated-patch configuration of the patch-clamp technique. Hence membrane depolarization and opening of the L-type voltage-gated Ca2+ channels were responsible for the first transient in [Ca2+]i. The second slower increase in [Ca2+]i was prolonged but readily reversed by the disulphide-reducing agent 1,4-dithiothreitol. This increase in [Ca2+]i was not decreased by nimodipine or by omission of extracellular Ca2+, but was eliminated when the Ins(1,4,5)P3-sensitive Ca2+ pool was first depleted by carbachol. Ryanodine or its beta-alanyl analogue did not release Ca2+ from intracellular stores, and a high concentration of ryanodine did not inhibit Ca2+ release by 2,2'-DTDP. The disulphide compound suppressed glucose metabolism and decreased the mitochondrial inner-membrane potential. We conclude that thiol oxidation by 2,2'-DTDP affects Ca2+ homeostasis in beta-cells by multiple mechanisms. However, unlike the situation in muscle, in beta-cells 2,2'-DTDP releases Ca2+ from intracellular pools by mechanisms that do not involve activation of ryanodine receptors. Instead, in these cells the Ins(1,4,5)P3-sensitive intracellular Ca2+ store comprises an alternative target for the Ca(2+)-mobilizing action of the reactive disulphide compound.

Nitric oxide induces intracellular Ca2+ mobilization and increases secretion of incorporated 5-hydroxytryptamine in rat pancreatic β-cells

This study is the first to demonstrate that low concentrations of aqueous NO induce intracellular Ca2+ mobilization and an increase in secretory activity of rat pancreatic β-cells. Application of NO solution (2 μM) resulted in a transient increase in the free intracellular Ca2+ concentration ([Ca2+]i) of isolated cells, as assessed by video ratio imaging and single wavelength microfluorimetry. Amperometry revealed a simultaneous increase in the release of preloaded 5-hydroxytryptamine from the isolated cells. The NO-induced Ca2+ response primarily involves mobilization of endoplasmic reticulum Ca2+ stores, since the response was retained when cells were transferred to low Ca2+ medium, and completely inhibited when cells were pretreated with 10 μM thapsigargin. The Ca2+ response was also inhibited when cells were incubated with a high concentration of ryanodine (200 μM), suggesting that Ca2+ mobilization is via a ryanodine-sensitive store.

Effect of ischemia and ischemia-reperfusion on ryanodine binding and Ca 2+ uptake of cardiac sarcoplasmic reticulum

The effect of 15 min of global, normothermic ischemia on 3H-ryanodine binding and the oxalate-supported Ca 2+ uptake of cardiac sarcoplasmic reticulum (SR) was investigated in parallel using ventricular homogenates of isolated perfused rat hearts. Ischemia increased the Ca 2+ efflux under the uptake assay conditions, as demonstrated by the greater stimulation of Ca 2+ uptake by high concentrations of ryanodine (+RY) to close the SR Ca 2+ channel. This effect was partially reversed by reperfusion. Ischemia depressed Ca 2+ uptake rate -RY at free [Ca 2+] of 0.4 μ m and above, while the depression +RY was significant only above 10 μ m Ca 2+. We tested the hypothesis that inhibition of the Ca-ATPase alone. by adding thapsigargin or cyclopiazonic acid, could reproduce the effects of ischemia on the homogenate Ca 2+ uptake rate. Thapsigargin or cyclopiazonic acid proportionally depressed Ca 2+ uptake rate +RY and -RY and produced distinctly different effects of ischemia. Ischemia did not change the B max or K d for equilibrium 3H-ryanodine binding, or the Hill coefficient or K Ca for the [Ca 2+]-dependence of equilibrium 3H-ryanodine binding. The rate of ryanodine binding. measured under the uptake conditions, was increased by ischemia and further increased by reperfusion. The effect of ischemia on the rate and extent of equilibrium binding to the high-affinity ryanodine binding site were unrelated to the highly reproducible effects on SR Ca 2+ uptake rates measured in the homogenate.

Modulations of membrane potential oscillations with drive, calcium overload, ryanodine, and caffeine

The objective of this study is to determine the conditions that generate overdrive excitation and overdrive suppression in canine cardiac Purkinje tissue superfused in vitro. Drive-induced (3 Hz) perturbations in the membrane potential of a calcium overloaded (induced by strophanthidin) Purkinje fiber (from a canine heart) were differently modulated by caffeine and ryanodine. Whereas the postdrive oscillations in the membrane potential V os (single or multiple oscillations in the diastole of the action potential) and/or spontaneous rate (postdrive suppression or postdrive excitation [PDE]) depended on the concentration of strophanthidin (PDE occurred at 2.5 × 10 −7 M, and V os were seen variably at several concentrations), caffeine (2–3 mM) in the presence of a lower concentration of strophanthidin (1.25 × 10 −7 M) induced PDE. At these lower concentrations, either drug administered alone only induced V os. On the contrary, the characteristic effects of ryanodine (10 −8 M) in the presence of strophanthidin (2.5 × 10 −7 M) were either a consistent postdrive suppression immediately or the induction of a pronounced afterdepolarization ([AD] a depolarization following the repolarization of the action potential) whose amplitude decreased with time and suppression. At higher concentrations of ryanodine (10 −5 M-10 −6 M) in a calcium overloaded tissue (strophanthidin, 1.25 × 10 −7 M) overdrive induced a pronounced AD in most cases, with subsequent depolarization and cessation of activity in less than 20 minutes. Ryanodine alone caused suppression of postdrive diastolic potential at lower concentrations (10 −9 M-10 −8 M), a pronounced AD (amplitude diminished with later drives), and suppression at higher concentrations (10 −6 M-10 −5 M). The tissue eventually depolarized (at higher concentrations) and ceased all activity. The authors conclude that caffeine (2–3 mM) had synergistic effects on the already-present calcium overload and, hence, induced PDE. Ryanodine, by its effects on either the sarcoplasmic reticulum and/or plasma membrane, may have reduced K + conductance nonspecifically in countering PDE without affecting the occurrence of calcium overload in this tissue.

Effect of low perfusate [Ca2+] and diltiazem on cardiac sarcoplasmic reticulum in myocardial stunning

The ability of low perfusate Ca2+ concentration ([Ca2+]) or diltiazem to improve sarcoplasmic reticulum (SR) function and mechanical performance after ischemia-reperfusion was investigated using an isovolumic Langendorff preparation. SR function was evaluated by the oxalate-supported Ca2+ uptake rates of ventricular homogenates. Influx of Ca2+ was estimated from the rate of Ca2+ uptake in the presence of high concentrations of ryanodine (500 microM) to close the Ca2+ efflux channel. Ca2+ efflux under the assay conditions was estimated as the difference in Ca2+ uptake rate in the presence and absence of ryanodine. Ten and fifteen min of global, normothermic ischemia decreased the Ca2+ uptake rate in the presence of ryanodine, suggesting that Ca2+ influx was decreased. The effect of ischemia on Ca2+ influx was not altered by preperfusion with low [Ca2+] (0.2 mM) or with 1.0 microM diltiazem. Ischemia decreased SR Ca2+ uptake rate twice as much in the absence of ryanodine as in its presence, indicating an increased efflux of Ca2+. This increased efflux was reduced by preperfusion with either low [Ca2+] or diltiazem. The decreased Ca2+ influx was completely reversed by 15 min of reperfusion, whereas the increased Ca2+ efflux was only partially reversed. These results indicate that ischemia exerts independent effects on the SR Ca2+ influx and efflux pathways. The results also suggest that one site of cardiac protection by low [Ca2+] or diltiazem is the ryanodine-sensitive Ca2+ efflux pathway of the SR, rather than the Ca2+ influx pathway.

A discrete [formula omitted] exchange dependent, Ca 2+ compartment in cultured neonatal rat heart cells. Characteristics, localization and possible physiological function

A pool of calcium has been identified in cultured neonatal rat heart cells whose exchange is dependent on extracellular sodium and calcium. In the absence of extracellular sodium and calcium this pool retains at least 760 μmol calcium/kg dry weight, which is released upon the introduction of sodium and calcium, with a t 1 2 of 4.4 s. Video microscopy experiments of non-stimulated cells showed that the cells, despite the retention of 760 μmoles calcium/kg dry weight, do not develop contracture, indicating that the majority of the pool is not retained in the general cytoplasm. Neither is the pool displaced by lanthanum. Several probes for the sarcoplasmic reticulum were used. Ryanodine (1 μM) had no significant effect on the size of the pool. Caffeine reduced the size of the pool by 60% and thapsigargin, and inhibitor of the sarcoplasmic reticulum Ca-ATPase reduced the content of the pool by 70%. High concentrations of ryanodine (1 mM) reduced the pool even further. The experimental data, in association with recently developed concepts of the diadic region [1], indicate that the pool may reside in the sub-sarcolemmal space between the sarcoplasmic reticulum and the sub-sarcolemma. The physiological significance of this pool is discussed.

Digestion of cardiac and skeletal muscle junctional sarcoplasmic reticulum vesicles with calpain II. Effects on the Ca2+ release channel.

The Ca2+ release channel and ryanodine receptor are activities copurifying with the 400,000-450,000 Da high molecular weight protein of cardiac and skeletal junctional sarcoplasmic reticulum. Calpain II, an endogenous cytosolic protease, was used to selectively degrade the high molecular weight protein in cardiac and skeletal muscle sarcoplasmic reticulum vesicles, and its effects on the activity of the Ca2+ release channel and [3H]ryanodine binding sites were analyzed. Degradation of the high molecular weight protein was associated with appearance of 315,000 and 150,000 Da proteolytic fragments and with a change in the ultrastructure of the "feet," extravesicular projections that protrude from the junctional sarcoplasmic reticulum membrane. The maximal number of [3H]ryanodine binding sites and the affinities of the sites for ryanodine were not remarkably affected by calpain II. Ca2+ release channels recorded from nondegraded cardiac and skeletal membrane vesicle preparations had slope conductances of 85 and 110 pS, respectively, measured with 1 microM cis-Ca2+ and 50 mM trans-Ba2+. Proteolysis did not alter the unitary channel conductances but did increase the percentage of channel open times from 36% to more than 90%. After proteolysis, channel opening remained dependent on micromolar cis-Ca2+, and high concentrations of ryanodine (300 microM) still blocked the channel. Our results suggest that proteolysis of the Ca2+ release channel with calpain II selectively impairs its inactivation, leaving its unitary conductance and the requirement for micromolar Ca2+ intact.