Absence Of Ryanodine Research Articles

Mechanisms of the antispasmodic activity of 3,5-di- t-butyl catechol (DTCAT) on rat vascular smooth muscles

In skeletal muscle sarcoplasmic reticulum vesicles, 3,5-di- t-butyl catechol (DTCAT) promotes the release of Ca 2+ through the activation of ryanodine receptor Ca 2+ channels. DTCAT mechanical and electrophysiological effects have now been investigated in rat aorta rings and single tail artery myocytes. Rat aorta rings incubated with either 30 μM ryanodine or 100 μM DTCAT developed tension, which averaged 36% and 7%, respectively, of that induced by phenylephrine. DTCAT reduced concentration-dependently both aorta ring contractions to high K + (IC 50 = 13.5 μM) and L-type Ba 2+ current (IC 50 = 22.0 μM) in isolated myocytes. Tetraethylammonium did not reverse the antispasmodic effect of DTCAT in rings stimulated with either 25 or 60 mM K +. DTCAT relaxed concentration-dependently phenylephrine-pre-contracted rings with intact endothelium (IC 50 = 10.9 μM). This effect was markedly reduced by pre-incubation of rings with 100 μM Nω-nitro- l-arginine methyl ester. DTCAT antagonised phenylephrine-induced contractions in endothelium-deprived rings, either in the presence or in the absence of ryanodine (IC 50 = 18.7 μM and 39.8 μM, respectively). Furthermore, both DTCAT (IC 50 = 53.3 μM) and ryanodine reduced significantly the response to phenylephrine in the absence of extracellular Ca 2+. Phenylephrine-stimulated influx of extracellular Ca 2+ was markedly inhibited when tissues were pre-treated with DTCAT (IC 50 = 19.0 μM) as well as nifedipine. DTCAT (> 100 μM) was also able to antagonise the contractions induced by phorbol 12-myristate, 13-acetate. In conclusion, this is the first demonstration that DTCAT inhibits vascular smooth muscle voltage-operated Ca 2+ channels and promotes the release of endothelial nitric oxide. Ryanodine receptor Ca 2+ channels activation or the impairment of the contractile apparatus by DTCAT seem to play a secondary role in its vascular activity.

Tight coupling between the rate of rise of Ca 2+ transient and peak twitch contraction in guinea-pig papillary muscle

We evaluated the relationship between cytoplasmic Ca 2+ concentration ([Ca 2+] i) and force in guinea-pig papillary muscles loaded with a fluorescent Ca 2+ indicator, fura-PE3. In the absence of ryanodine, [Ca 2+] i transient and force were altered by changing extracellular Ca 2+ concentration and stimulation frequency, and also by adding methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)-pyridine-5-carboxylate (Bay K 8644) or ouabain. Under these conditions, the peak force correlated linearly with the maximal rate of rise of [Ca 2+] i ( γ=0.948) more than the peak [Ca 2+] i transient ( γ=0.737). Ryanodine inhibited the increase in the maximal rate of rise of [Ca 2+] i resulting in abolishment of the correlation between force and the maximal rate of rise of [Ca 2+] i. These results suggest that the maximal rate of rise of [Ca 2+] i reflects Ca 2+ release from the sarcoplasmic reticulum, and this fraction of [Ca 2+] i is crucial for determining the amplitude of twitch contractions when the sarcoplasmic reticulum is intact in guinea-pig papillary muscle.

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.

Effect of Ischemia on the Fraction of Ryanodine-sensitive Cardiac Sarcoplasmic Reticulum

The effect of 15 min of global, normothermic ischemia on cardiac sarcoplasmic reticulum (SR) was investigated using the Ca2+uptake rate and3H-ryanodine binding of ventricular homogenates and isolated SR vesicles. Ischemia did not affect ryanodine binding in the homogenate, while it increased it in the isolated SR vesicles. Although ischemia decreased the homogenate oxalate-supported Ca2+uptake rate, measured in the presence of high ryanodine to close the ryanodine-sensitive efflux pathway (+RY), its decrease of the Ca2+uptake rate, measured in the absence of ryanodine (−RY), was more marked. This finding was also observed in the isolated SR. Although inhibition of the Ca-ATPase and its coupled Ca2+uptake by thapsigargin proportionately decreased SR Ca2+uptake −RY and +RY, ischemia decreased the Ca2+uptake −RY proportionately more. This result suggested that there was a greater fraction of Ca2+uptake activity in ryanodine-sensitive vesicles after ischemia. However, ischemia also reduced the yield of SR activity in the isolated SR fraction and the results could potentially be due to differential selection of ryanodine-sensitive and ryanodine-insensitive SR in the isolation procedure. We directly tested the hypothesis that ischemia changes the fraction of Ca2+uptake activity in the ryanodine-sensitive vesicles by estimating the Ca-oxalate capacity measured +RY and −RY. Ischemia decreased the capacity −RY much more than +RY in the homogenate, indicating that more of the SR volume and Ca2+uptake activity was in the ryanodine-sensitive vesicles after ischemia.

Rapid, simple and sensitive microassay for skeletal muscle homogenates in the functional assessment of the Ca-release channel of sarcoplasmic reticulum: application to diagnosis of susceptibility to malignant hyperthermia.

A microassay is demonstrated for functional characterization of the Ca(2+)-release channel (CRC) of sarcoplasmic reticulum (SR) of skeletal muscle using swine with susceptibility to malignant hyperthermia (MH). Diluted muscle homogenates, indo-1 and ratiometric dual-emission spectrofluorometry are used to monitor Ca(2+)-lowering activity in real-time in the presence and absence of ryanodine at exposures that open and close the CRC. Reactions are initiated with 50 microM CaCl2 to raise ionized Ca2+ concentration near 1 microM and MgATP to activate the Ca(2+)-ATPase pump. Oxalate is included to precipitate Ca2+ within the SR. The assay requires less than 30 mg muscle, which may be cryopreserved, and is completed within 20 min of thawing the tissue. Maximum SR Ca(2+)-ATPase pumping and CRC activities, degree of CRC activation, and Ca(2+)-buffering capacity can be determined. Using this assay we studied muscle from MH-susceptible swine and demonstrated that whereas maximal Ca(2+)-ATPase pumping and CRC activities are normal, the CRC activity after addition of a bolus of Ca2+ is 50% greater in heterozygotes and 100% greater in homozygotes for the MH mutation. Hypersensitivity to CRC agonists, such as caffeine, and an associated hyposensitivity to CRC antagonists such as Mg2+ is also demonstrated. Genotypes for the MH mutation site can be discriminated from each other by determining Ca(2+)-lowering activities and the effect of ryanodine on them.

Effect of Ryanodine on Sarcoplasmic Reticulum Ca 2+ Accumulation in Nonfailing and Failing Human Myocardium

The purpose of this study was to determine whether abnormal Ca2+ release through ryanodine-sensitive Ca2+ channels in the sarcoplasmic reticulum might contribute to the abnormal [Ca2+]i homeostasis that has been described in failing human myocardium. Occupancy of low-affinity ryanodine binding sites on ryanodine-sensitive Ca2+ channels stimulates oxalate-supported, ATP-dependent Ca2+ accumulation in sarcoplasmic reticulum-derived microsomes by inhibiting concurrent Ca2+ efflux through these channels. We examined the effects of 0.5 mmol/L ryanodine on 45Ca2+ accumulation in microsomes prepared from nonfailing (n = 8) and failing (n = 10) human left ventricular myocardium. In the absence of ryanodine, 45Ca2+ accumulation reached similar levels in microsomes from nonfailing and failing hearts. Incubation with 0.5 mmol/L ryanodine caused a 52.2 +/- 6.5% increase in peak 45Ca2+ accumulation in microsomes from nonfailing hearts and a 24.3 +/- 4.1% increase in microsomes from failing hearts. The density of high-affinity ryanodine binding sites and the inhibition of [3H]ryanodine dissociation from these sites by 0.1 mmol/L ryanodine were similar in microsomes from nonfailing and failing hearts. These results, which demonstrate a diminished stimulation of Ca2+ accumulation by ryanodine in sarcoplasmic reticulum-derived microsomes from failing human myocardium that could be explained by an uncoupling of the occupancy of low-affinity ryanodine binding sites from the reduction in the open probability of these channels or by concurrent Ca2+ efflux through a ryanodine-insensitive mechanism, are evidence that increased efflux of Ca2+ from the sarcoplasmic reticulum may contribute to the abnormal [Ca2+]i homeostasis described in failing human myocardium.

Desensitization of the skeletal muscle ryanodine receptor: evidence for heterogeneity of calcium release channels

Ca release channels from the junctional sarcoplasmic reticulum (SR) membranes of rabbit skeletal muscle were incorporated into the lipid bilayer membrane, and the inactivation kinetics of the channel were studied at large membrane potentials. The channels conducting Cs currents exhibited a characteristic desensitization that is both ligand and voltage dependent: 1) with a test pulse to -100 mV (myoplasmic minus luminal SR), the channel inactivated with a time constant of 3.9 s; 2) the inactivation had an asymmetric voltage dependence; it was only observed at voltages more negative than -80 mV; and 3) repetitive tests to -100 mV usually led to immobilization of the channel, which could be recovered by a conditioning pulse to positive voltages. The apparent desensitization was seen in approximately 50% of the experiments, with both the native Ca release channel (in the absence of ryanodine) and the ryanodine-activated channel (1 microM ryanodine). The native Ca release channels revealed heterogeneous gating with regard to activation by ATP and binding to ryanodine. Most channels had high affinity to ATP activation (average open probability (po) = 0.55, 2 mM ATP, 100 microM Ca), whereas a small portion of channels had low affinity to ATP activation (po = 0.11, 2 mM ATP, 100 microM Ca), and some channels bound ryanodine faster (< 2 min), whereas others bound much slower (> 20 min). The faster ryanodine-binding channels always desensitized at large negative voltages, whereas those that bound slowly did not show apparent desensitization. The heterogeneity of the reconstituted Ca release channels is likely due to the regulatory roles of other junctional SR membrane proteins on the Ca release channel.

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.

Effects of inhibition of transsarcolemmal calcium influx by nickel on force of postrest contraction and on contracture induced by rapid cooling

The aim was to characterise the influence of a decrease in transsarcolemmal calcium influx on the calcium content of the sarcoplasmic reticulum and on the force of postrest contraction, which is largely activated by calcium released from the sarcoplasmic reticulum. Isometric force of postrest contractions in isolated rabbit papillary muscles was measured in the absence and in the presence of ryanodine (1 microM). The force measured in the presence of ryanodine was used as an index of transsarcolemmal calcium influx. The calcium content of the sarcoplasmic reticulum was estimated by rapid cooling induced contracture measurements. Nickel (Ni2+) was used to decrease the calcium influx, and the results were compared to those obtained by lowering the extracellular calcium concentration. The addition of Ni2+ or lowering extracellular Ca2+ markedly decreased the force of postrest contraction measured in the presence of ryanodine, and moderately decreased the force of postrest contraction measured in the absence of ryanodine. Neither the addition of Ni2+ nor lowering extracellular Ca2+ decreased maximum rapid cooling induced contracture; this was actually increased in the presence of Ni2+ even though a larger number of stimuli was required in the presence than in the absence of Ni2+ for the force of the subsequently elicited contracture to reach the maximum level. The results suggest: (1) that a decrease in the calcium influx reduces the force of the postrest contraction by decreasing the amount of calcium required to trigger the release of calcium from the sarcoplasmic reticulum, without decreasing its maximum calcium content; and (2) that Ni2+ decreases the rate of filling of sarcoplasmic reticular calcium stores even though the calcium content of the sarcoplasmic reticulum after a sufficiently large number of beats is higher in the presence of Ni2+ than in its absence.

Effects of local anesthetics on single channel behavior of skeletal muscle calcium release channel.

The effects of the two local anesthetics tetracaine and procaine and a quaternary amine derivative of lidocaine, QX314, on sarcoplasmic reticulum (SR) Ca2+ release have been examined by incorporating the purified rabbit skeletal muscle Ca2+ release channel complex into planar lipid bilayers. Recordings of potassium ion currents through single channels showed that Ca(2+)- and ATP-gated channel activity was reduced by the addition of the tertiary amines tetracaine and procaine to the cis (cytoplasmic side of SR membrane) or trans (SR lumenal) side of the bilayer. Channel open probability was lowered twofold at tetracaine and procaine concentrations of approximately 150 microM and 4 mM, respectively. Hill coefficients of 2.0 and greater indicated that the two drugs inhibited channel activity by binding to two or more cooperatively interacting sites. Unitary conductance of the K(+)-conducting channel was not changed by 1 mM tetracaine in the cis and trans chambers. In contrast, cis millimolar concentrations of the quaternary amine QX314 induced a fast blocking effect at positive holding potentials without an apparent change in channel open probability. A voltage-dependent block was observed at high concentrations (millimolar) of tetracaine, procaine, and QX314 in the presence of 2 microM ryanodine which induced the formation of a long open subconductance. Vesicle-45Ca2+ ion flux measurements also indicated an inhibition of the SR Ca2+ release channel by tetracaine and procaine. These results indicate that local anesthetics bind to two or more cooperatively interacting high-affinity regulatory sites of the Ca2+ release channel in or close to the SR membrane. Voltage-dependent blockade of the channel by QX314 in the absence of ryanodine, and by QX314, procaine and tetracaine in the presence of ryanodine, indicated one low-affinity site within the conduction pathway of the channel. Our results further suggest that tetracaine and procaine may primarily inhibit excitation-contraction coupling in skeletal muscle by binding to the high-affinity, regulatory sites of the SR Ca2+ release channel.

Effect of perfusate [Ca2+] on cardiac sarcoplasmic reticulum Ca2+ release channel in isolated rat hearts.

The effect of perfusate [Ca2+] on the function of cardiac sarcoplasmic reticulum (CSR) was assessed by the oxalate-supported Ca2+ uptake rate of ventricular homogenates of isolated rat hearts maintained in a modified Langendorff preparation. The total Ca2+ pumping activity of the CSR was determined by using 20 microM ruthenium red or 625 microM ryanodine to close the CSR Ca2+ release channel. The homogenate Ca2+ uptake rate in the absence of ruthenium red or ryanodine decreased progressively with increasing perfusate [Ca2+] (25.7 +/- 1.2, 21.4 +/- 1.5, 17.2 +/- 1.1, and 16.3 +/- 1.2 [mean +/- SEM] nmol Ca2+.min-1.mg-1 for hearts perfused for 5 minutes with 0.2, 1.4, 2.8, and 5.6 mM Ca2+, respectively; p = 0.0001; n = 8). This depression was not observed when Ca2+ uptake was assayed in the presence of ryanodine or ruthenium red. Since the Ca2+ uptake in the presence of ryanodine or ruthenium red is determined by the Ca(2+)-ATPase, this result suggests that perfusion with varying [Ca2+] did not affect the Ca(2+)-ATPase. The observed decrease in Ca2+ uptake in the absence of ryanodine or ruthenium red is caused by an increased efflux through the ryanodine-sensitive Ca2+ release channel. When hearts perfused for 5 minutes with 0.2 or 5.6 mM Ca2+ were reperfused for 10 minutes with 1.4 mM Ca2+, homogenate Ca2+ uptake rates were restored to near control levels. These effects of perfusate Ca2+ were not direct effects, because changes in the [Ca2+] of the homogenization medium did not alter the homogenate Ca2+ uptake activity in either the presence or absence of ryanodine. The homogenate Ca2+ uptake rates were unaffected by prior active loading of the CSR with Ca2+. These results suggest a regulatory role of perfusate Ca2+ in increasing the open state of the ryanodine-sensitive Ca2+ release channel that is distinct from the beat-to-beat regulation of Ca2+ release from the CSR by Ca2+ (Ca(2+)-induced Ca2+ release).

The Influence of Temperature on Ryanodine Sensitivity and the Force-Frequency Relationship in the Myocardium of Rainbow Trout

The relationship between stimulation frequency and contraction was established for ventricular strips from rainbow trout heart at 5, 15 and 25 degrees C. Compared to mammalian species, changes in temperature had little impact on force development in trout ventricle at physiologically relevant stimulation frequencies. However, the force-frequency relationship was changed from a biphasic response with a minimum around 0.2 Hz at 5 and 15 degrees C to a monophasic decline in force with increasing frequency at 25 degrees C. Ryanodine reversed the negative force-frequency relationship at 25 degrees C. Potentiation of twitch force after a 5 min rest period was increased from 121 +/- 4% at 15 degrees C to 209 +/- 12% at 25 degrees C. A similar augmentation was seen for the maximal rate of force development. Rest potentiation of both force and maximal rate of force development (dF/dT) was abolished by ryanodine at both 15 and 25 degrees C. The ryanodine concentration causing a half-maximal reduction in rest potentiation of force was 51 nmol l-1 at 25 degrees C and 483 nmol l-1 at 15 degrees C. Rest potentiation was maximally reduced by 10 mumol l-1 ryanodine to 50 and 79% of the value in the absence of ryanodine at 25 and 15 degrees C, respectively. At 5 degrees C, rest potentiation was similar to that at 15 degrees C. At 5 degrees C, there was no rest potentiation of dF/dT and ryanodine did not reduce rest potentiation of force. Instead, rest potentiation was correlated with a potentiation of time to peak tension (TPT) at 5 degrees C. Thus, in trout ventricle, force correlates with TPT at 5 degrees C and seems to be regulated by a ryanodine-insensitive mechanism, while at 25 degrees C force is correlated with the maximal rate of force development and the sarcoplasmic reticulum appears to contribute significantly to excitation-contraction coupling.

Interdependence of ryanodine binding, oligomeric receptor interactions, and Ca 2+ release regulation in junctional sarcoplasmic reticulum

We have examined ryanodine binding to its receptor (RR) and compared its effect on Ca 2+ release to the Ca 2+ release triggered by Ca 2+ plus ATP, using vesicular fragments of junctional terminal cisternae (JTC) obtained from skeletal muscle. Ryanodine binding is slow (taking hours or days to complete) and is highly temperature ( Q 10 = 4) and Ca 2+ dependent. At equilibrium, the extent of binding increases as the concentration of ryanodine is raised above 10 −9 m, exhibiting negative cooperativity and reaching the stoichiometry of the 560,000-Da RR chains near 10 −5 m ryanodine. The specificity of the high affinity binding is demonstrated by competitive binding of ryanodine analogs. Kinetic studies using rapid filtration show that, in the absence of ryanodine, rapid ( k = 15 s −1) release of Ca 2+ follows a triggering exposure of loaded JTC vesicles to perfusion media containing Ca 2+ plus ATP. Induction of this release has no lag period and displays minimal temperature dependence. In contrast, prolonged exposure of JTC vesicles to low (10 −7 m) ryanodine concentrations changes the JTC to a state permitting slow ( k = 1 s −1) release of Ca 2+ even in the absence of the Ca 2+ plus ATP trigger. Higher (>μ m) concentrations of ryanodine do not allow any Ca 2+ release and prevent even the release normally triggered by Ca 2+ plus ATP. Our data suggest that ryanodine binds to the open state of the tetrameric RR, inducing protein conformational changes and altered oligomeric interactions. Binding of the first molecule of ryanodine to one of the four binding sites on the receptor produces a partially closed and low conductance state of the Ca 2+ release channel and reduces the ryanodine binding affinity of the remaining sites. Ryanodine occupancy of all four binding sites on the receptor completes closure of the Ca 2+ channel and blocks the triggering action of Ca 2+ plus ATP. The tetrameric association of the RR chains is demonstrated by crosslinking with bifunctional reagents, generating crosslinked tetramers that retain ryanodine binding and Ca 2+ release functions.

Effect of external Ca2+ concentration on stretch-augmented natriuretic peptide secretion by rat atria

An in vitro noncontracting rat atrial preparation stretched at 37 degrees C by a distending pressure of 5.1 mmHg was used to examine effects of external Ca2+ concentration ([Ca2+]out, 0.05-3.0 mM) on secretion of immunoreactive atrial natriuretic peptide (ANP) in presence of saxitoxin (STX) and in presence or absence of ryanodine. Under these conditions, the time course of the amount (y) of ANP secreted per milligram dry atrium during 44 min could be approximated by a rate coefficient (k) according to the relation y = s[1 - e(-kt)], where s is the maximal amount secreted after a long time (t). Although k, the rate coefficient for stretch-augmented secretion, increased significantly as [Ca2+]out was raised, secretion inactivated progressively in a time- and [Ca2+]out-dependent manner. This time-dependent decrease was not prevented by ryanodine. We conclude that a component of ANP secreted by quiescent atria in vitro is positively modulated by [Ca2+]out and does not require ryanodine-sensitive Ca2+ release from sarcoplasmic reticulum. The [Ca2+]out-sensitive processes underlying time-dependent inactivation of secretion remain undetermined.

Differential Modulation of Norepinephrine-Induced Contractile Response by Ryanodine and Verapamil in the Isolated Aortic Ring of Spontaneously Hypertensive and Wistar-Kyoto Rats

Using ryanodine and verapamil, we compared the relative contributions of SR Ca2+ release and gated Ca2+ entry in arterial contractions induced by norepinephrine (NE) between spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY). Aortic rings of 10 SHR and 10 WKY aged 10-12 weeks were superfused in small water-jacketed tissue chambers with physiological salt solution and isometric tension was measured. The inhibition of the NE (3 x 10(-8) M)-induced contraction of aortic rings by ryanodine (10 microM) was significantly greater in WKY (40.1 +/- 6.9%) than in SHR (2.2 +/- 9.0%) (p less than 0.01). The inhibition of the NE-induced contraction by verapamil (10 microM) in the presence or absence of ryanodine (10 microM) was significantly greater in SHR than in WKY. The residual ryanodine and verapamil-insensitive component of NE-induced contraction was significantly greater in WKY than in SHR. Caffeine (5 mM)-induced contraction in the presence of verapamil and phentolamine was significantly smaller in SHR than in WKY. These results suggest that gated Ca2+ entry plays a more important role in Ca2+ control and that ryanodine-sensitive Ca2+ store is smaller, or the ryanodine receptor is altered in these tissues of SHR compared with those of WKY.

Ryanodine facilitates calcium‐dependent release of transmitter at mouse neuromuscular junctions

1. Quantal release of transmitter was measured intracellularly at mouse neuromuscular junctions in the presence and absence of ryanodine (Rnd). 2. Rnd at concentrations up to 1 microM did not significantly alter the frequency of miniature endplate potentials (m.e.p.ps) in the presence or absence of Ca2+, suggesting that Rnd is unlikely to alter the internal concentration of Ca2+ ([Ca2+]i) at rest. 3. In a high-K+ (10 mM) bathing solution, Rnd further potentiated the facilitatory effect of Ca2+ on the frequency (F, s-1) of m.e.p.ps. Rnd shifted the relationship between 1n(F) and 1n[Ca2+]o to lower concentrations. 4. In a high-Mg2+ bathing solution, Rnd did not affect the frequency of m.e.p.ps at any value of [Ca2+]o. However, Rnd slightly but significantly increased the quantal content (m) of e.p.ps. It shifted the relationship between 1n(m) and 1n[Ca2+]o to lower concentrations. These results suggest that Rnd potentiates the quantal release of transmitter after depolarization of the membrane or nerve impulse, in keeping with the cooperativity of Ca2+ at the active site. 5. A series of two closely spaced nerve impulses produced a facilitation of transmitter release, as judged by the quantal content (m2) of the second response in relation to that of the first one (m1), m2/m1. Rnd did not change the ratio m2/m1. Thus Rnd is unlikely to affect the rapid phase of the sequestration of Ca2+ inside the nerve terminal. 6. High levels of K+ (5 mM) and caffeine (2 mM) potentiated both modes of transmitter release, in a manner dependent on [Ca2+]o. Caffeine did not potentiate facilitation of transmitter release. 7. These results indicate that Rnd facilitates the quantal release of transmitter presumably via an increase in [Ca2 ]i by a manner different from that of high-K+ or caffeine. The results suggest that Rnd probably affects calcium turnover in neuronal cells.

Direct Effect of Halothane and Isoflurane on the Function of the Sarcoplasmic Reticulum in Intact Rabbit Atria

The negative inotropic effect of halothane and isoflurane on potentiated-state contractions of isolated rabbit atria in a normal Ca2+ (2.5 mM) medium was compared with the force depression in low Ca2+ media without an anesthetic. When this comparison was made in the presence of 1 microM ryanodine so that the force of contraction was dependent only upon transsarcolemmal Ca2+ influx with no Ca2+ contribution from the sarcoplasmic reticulum (SR), the force of contraction was depressed equally by 0.6% halothane in a normal Ca2+ medium and by a 1.5 mM Ca2+ medium without the anesthetic. Similarly, 1.0% halothane or 1.5% isoflurane and a 1.0 mM Ca2+ medium were equally depressant as were 2.4% isoflurane and a 0.5 mM Ca2+ medium. In the absence of ryanodine, where the atrial contractile activity is largely dependent on Ca2+ released from the SR, 0.6% halothane in the normal Ca2+ medium depressed contractile force by 32%, whereas the force was depressed by only 16% in the 1.5 mM Ca2+ medium without the anesthetic. Similar results were obtained when the effects of 1.0% halothane and of 1.0 mM Ca2+ were compared. In contrast, the force of contraction measured in the absence of ryanodine was not at all inhibited by 1.5% isoflurane and minimally (11%) inhibited by 2.4% isoflurane. Consequently, the force depression by isoflurane was less than that found in the low Ca2+ media.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of ryanodine on neonatal and adult rat heart: Developmental increase in sarcoplasmic reticulum function

Negative inotropic responses to nicardipine, MnCl 2 and ryanodine of isolated papillary muscles were compared between 1-day-old neonatal and adult rats. All of the drugs produced dose-dependent negative inotropic responses at both ages. Nicardipine and MnCl 2 were effective at lower concentrations in the neonate when compared to the adult, while ryanodine was more effective in the adult. Attenuation of the positive inotropic effect of isoproterenol by nicardipine was greater in the neonate, while that by ryanodine was greater in the adult. The relaxation phase of contraction was shortened by isoproterenol and prolonged by ryanodine in adult preparations, but there were no changes in the neonatal preparations. In the adult, increasing the stimulus frequency resulted in a decrease in contractile force (negative force-frequency relationship) at control conditions, while it produced rather slight increases in contractile force in the presence of ryanodine. In the neonate, increases in the stimulus frequency resulted in an increase in contractile force (positive force-frequency relationship) both in the presence and absence of ryanodine. It was concluded that contraction of the adult rat myocardium is highly dependent on Ca 2+ release from the sarcoplasmic reticulum, while that of the neonatal myocardium is more dependent on trans-sarcolemmal Ca 2+ influx. It was also speculated that the negative force-frequency relationship is produced by some factor(s) closely related to sarcoplasmic reticulum function.

Effects of ryanodine on skinned skeletal muscle fibers of the rabbit

The mechanism(s) of ryanodine-induced contracture of skeletal muscle were studied in skinned fibers from soleus (SL) and adductor magnus (AM) (slow- and fast-twitch skeletal muscles) of rabbits. Pieces of SL or AM were homogenized (sarcolemma disrupted). Single fibers were dissected from the homogenate and mounted on photodiode force transducers. At concentrations 1-50 microM, ryanodine slightly but significantly increased the submaximal Ca2+-activated tension development of the contractile proteins in skinned fibers of AM but not of SL. Ryanodine in uptake phase or release phase increased caffeine-induced tension transients in the SR of both muscle types; however, no dose-response relation was found. Ryanodine greater than or equal to 1 microM decreased, however, the second control tension transients in a dose-dependent manner. The depression was nearly irreversible and "activity"-dependent. The concentrations of ryanodine that inhibited the second control tension transients by 50% were 10 microM and 5 microM for SL and AM, respectively, following ryanodine administration in the release phase, and 100 microM and 30 microM, respectively, for these preparations after the drug was present in the uptake phase. The quantity of calcium released from the SR by Triton X-100 and caffeine in the second control tension transient was unchanged by ryanodine at all concentrations tested when compared with that of the absence of ryanodine. The present findings suggest that the ability of ryanodine to increase immediate calcium release from the SR, and in AM but not SL, to increase the sensitivity of the contractile proteins to Ca2+ underlies the contracture caused by this agent in intact skeletal muscles.(ABSTRACT TRUNCATED AT 250 WORDS)

Excitation-contraction coupling in myocardium of nonhibernating and hibernating chipmunks: effects of isoprenaline, a high calcium medium, and ryanodine.

The electromechanical responses to isoprenaline and a high calcium medium on cardiac muscles from nonhibernating and hibernating chipmunks were studied in the presence and the absence of ryanodine. In nonhibernating animal preparations, isoprenaline (5 X 10(-8) M) caused a marked positive inotropic effect with an increase in the amplitude of the action potential plateau and augmented the slow action potential in muscle depolarized with 26 mM K+. In hibernating animal preparations, isoprenaline failed to cause a positive inotropic effect in spite of an increase in the amplitude of action potential plateau and slow action potentials. Similar inotropic effects were caused in the two preparations when extracellular calcium was raised to 6 mM, but action potential plateau and slow action potentials of the two preparations were less affected by this procedure; only in nonhibernating animal preparations was the amplitude of the slow action potential slightly increased. Ryanodine (2 X 10(-6) M) partially inhibited the contraction but augmented the action potential plateau and slow action potentials in nonhibernating animal preparations, while in hibernating animal preparations, it eliminated the contraction and severely inhibited the action potential plateau and slow action potentials. The electrical effects of isoprenaline on the two preparations and of a high calcium medium on nonhibernating animal preparations were more pronounced in the presence of ryanodine than in the absence of it. However, the electrical activity on hibernating animal preparations was unaffected by a high calcium medium in either the presence or absence of ryanodine.(ABSTRACT TRUNCATED AT 250 WORDS)