3H-ryanodine Binding Research Articles

Bile acids activate ryanodine receptors in pancreatic acinar cells via a direct allosteric mechanism

The earliest critical event of pancreatitis is a long lasting high amplitude rise of intracellular Ca2+ concentration of the acinar cell, which can be triggered by high concentration of bile acids. Although, Ca2+-release through ryanodine receptors (RyR) is involved in the process, the significance and the exact mechanism of bile acid's action on RyR has not been fully elucidated yet. Therefore, we aimed to test with various techniques and aspects whether bile acids exert a direct effect on RyR and SERCA pump.Our data show that taurocholic acid (TCA)-induced Ca2+ release in pancreatic acinar cells was significantly reduced by the RyR antagonist dantrolene. Further, we show that TCA enhanced RyR's 3H-ryanodine binding and triggered robust Ca2+-release from RyR-enriched vesicles in the pathologically relevant concentration range. RyR single channel current analysis demonstrated that 200μM TCA induced a 5-fold increase in the channel's open probability and caused a significant lengthening of the mean open time. TCA also suppressed Ca2+-uptake rate and ATP-ase activity of SERCA-enriched vesicles, but interestingly, failed to decrease Ca2+ elimination rate in intact cells.Overall, our results strongly suggest that TCA opens RyR by an allosteric mechanism, which contribute significantly to bile acid-induced pathologic Ca2+-leak from the endoplasmic reticulum in pancreatic acinar cells.

Role of C-Term Tail of DHPR β1A in the DHPR/RyR1 Interaction

Although the mechanism by which the DHPR β1a subunit supports EC-coupling is still debatable it is apparent that C-terminal domain of β1a (β-Ct) is an intricate component of the interaction between the DHPR complex and RyR1. To characterize the molecular components of β-Ct involved in DHPR/RyR1 signaling we tested the effect of progressive truncation of β1a subunit on EC-coupling and RyR1 activity. To do this cDNA constructs carrying truncations of either the 52 (β-52), 38 (β-38) or 14 (β-14) most C-terminal amino acid residues of β1a were expressed in β-null myotubes and then tested for their ability to restore depolarization-induced Ca2+ release in Fluo-4 loaded cells. Whereas β-null myotubes expressing constructs β-52 and β-38 were unresponsive to K+ depolarization the cells expressing β-14 displayed EC-coupling that was indistinguishable from that of cells expressing wt-β1a (K+EC50: β1a = 23mM, β-14 =21mM). Thus, these results identify a segment of β-Ct of up to 24 amino acids that appears to be critical for the functional interaction between DHPR and RyR1 during EC-coupling. To test for specific interactions between the β-Ct and RyR1 we then studied the effect of purified β subunits on RyR1 activity. Using 3H-ryanodine binding to SR vesicles from skeletal muscle we found that purified wt-β1a subunit had an inhibitory effect in RyR1 activity in a dose-dependent manner (IC50=38nM). However, purified β-38, a construct that failed to restore EC-coupling in myotubes, failed to inhibit 3H-ryanodine binding to RyR1. Overall, these results are consistent with the hypothesis that DHPR β1a subunit functionally interacts with RyR1 through a domain in the final 38 amino acids in its C-terminal tail.Supported by NIH Grants 5K01AR054818-02 (to CFP) and 1P01AR044750 (to PDA)

Effects of power frequency magnetic field on Ca2+ transport of skeletal muscle sarcoplasmic reticulum vesicles

To investigate the effects of power frequency magnetic field on the Ca2+ transport dynamics of isolated sarcoplasmic reticulum vesicles. The assays of Ca2+ uptake time course and the Ca2+-ATPase activity of sarcoplasmic reticulum vesicles were investigated by using dynamic mode of spectrometry with a Ca2+ dye; Ca2+ release channel activation was examined by 3H-ryanodine binding and Ca2+ release assays; membrane fluidity of sarcoplasmic reticulum vesicles was examined by fluorescence polarization, without or with exposure to the vesicles at a 0.4 mT, 50 Hz sinusoidal magnetic field. 0.4 mT, 50 Hz sinusoidal magnetic field exposure caused about a 16% decline of the initial Ca2+ uptake rate from a (29.18 +/- 3.90) pmol.mg(-1).s(-1) to a (24.60 +/- 3.81) pmol.mg(-1).s(-1) and a 26% decline of the Ca2+-ATPase activity from (0.93 +/- 0.05) micromol.mg(-1).min(-1) to (0.69 +/- 0.07) micromol.mg(-1).min(-1) of sarcoplasmic reticulum vesicles, whereas caused a 15% increase of the initial Ca2+ release rate from (4.83 +/- 0.82) pmol.mg(-1).s(-1) to (5.65 +/- 0.43) pmol.mg(-1).s(-1) and a 5% increase in 3H-ryanodine binding to the receptor from (1.10 +/- 0.12) pmol/mg to (1.16 +/- 0.13) pmol/mg, respectively. The decline of Ca2+-ATPase activity and the increase of Ca2+ release channel activity should result in a down-regulation of Ca2+ dynamic uptake and an up-regulation of Ca2+ release induced by exposing the sarcoplasmic reticulum to a 0.4 mT, 50 Hz power frequency magnetic field.

A Ca 2+-Binding Domain in RyR1 that Interacts with the Calmodulin Binding Site and Modulates Channel Activity

A fragment of RyR1 (amino acids 4064–4210) is predicted to fold to at least one lobe of calmodulin and to bind Ca 2+. This fragment of RyR1 (R4064–4210) was subcloned, expressed, refolded, and purified. Consistent with the predicted folding pattern, R4064–4210 was found to bind two molecules of Ca 2+ and undergo a structural change upon binding Ca 2+ that exposes hydrophobic amino acids. R4064–4210 also binds to RyR1, the L-type Ca 2+ channel (Cav 1.1), and several synthetic calmodulin binding peptides. Both R4064–4210 and a peptide representing the calmodulin-binding region of RyR1 (R3614–3643) alter the Ca 2+ dependence of ( 3H)ryanodine binding to RyR1, suggesting that they may both be interfering with an intramolecular interaction between amino acids 4064–4210 and amino acids 3614–3643 in the native RyR1 to alter or regulate the response of the channel to changes in Ca 2+ concentration. The finding that a domain within RyR1 binds Ca 2+ and interacts with calmodulin-binding motifs may provide insights into the mechanism for calcium- and calmodulin-dependent regulation of this channel and perhaps for its regulation by the L-type Ca 2+ channel.

Evaluation of indices of skeletal muscle contraction in areas of referred hyperalgesia from an artificial ureteric stone in rats

This study examined indices of skeletal muscle contraction in a rat model of referred muscle hyperalgesia from artificial ureteric calculosis [left oblique muscle (OE) for ipsilateral stone]. In specimens from the left versus right OE of stone-implanted female rats, a significant increase was found in membrane fluidity ( P<0.01) and Ca 2+-ATPase activity ( P<0.0001) and a significant decrease in 3H-ryanodine binding ( P<0.0001) and in I band length/sarcomere length ratio (contraction index) ( P<0.01). The increase in Ca 2+-ATPase activity was directly and significantly related to the number of rats’ ureteral ‘crises’ ( P<0.02). The results indicate a state of contraction in the hyperalgesic muscle, whose extent correlates to the algogenic activity of the ureteral stone.

Altered cellular calcium regulatory systems in a rat model of cirrhotic cardiomyopathy

Background & Aims: Decreased cardiac contractility has been observed in cirrhosis, but the cause remains unclear. Because cardiomyocyte contraction depends on Ca2+ influx entering via L-type Ca2+ channels (ICa,Ls) to activate Ca2+ release from the sarcoplasmic reticulum, we postulated that the Ca2+ transients may be abnormal in cirrhotic cardiomyocytes. We aimed to investigate the status of the cellular Ca2+-regulatory system in a rat model of cirrhotic cardiomyopathy. Methods: Cirrhosis was induced by bile duct ligation. The ICa,L protein expression was detected by Western blotting. Ca2+ currents were measured electrophysiologically. The intracellular Ca2+ system, which includes the ryanodine receptor 2 (RYR2), sarcoplasmic reticulum Ca2+-pump adenosine triphosphatase (SERCA2), and Ca2+-binding protein were quantitatively assayed by reverse-transcription polymerase chain reaction and Western blots and functionally by 3H-ryanodine binding and radiolabeled Ca2+ uptake. Results: ICa,L protein expression was reduced in cirrhotic rats compared with controls, and the peak inward Ca2+ current was significantly less. At all membrane potentials examined, ICa,Ls current densities from cirrhotic animals were consistently lower, and the response to maximal isoproterenol stimulation was also significantly lower. Protein expression and messenger RNA transcription for RYR2, SERCA2, and calsequestrin were quantitatively unchanged, and 3H-ryanodine binding characteristics and Ca2+ uptake were also unaltered. Conclusions: We conclude that the decreased cardiac contractility in cirrhotic cardiomyocytes is caused by dysfunction of the Ca2+-regulatory system. Plasma membrane ICa,Ls are quantitatively reduced and functionally depressed, whereas intracellular systems are intact.GASTROENTEROLOGY 2001;121:1209-1218

Calcium uptake, release and ryanodine binding in melanosomes from retinal pigment epithelium

High levels of calcium have been reported in pigmented tissues of the vertebrate eye, such as retinal pigment epithelium (RPE). Melanin granules also have high calcium concentrations, suggesting that melanin granules may be a calcium reservoir. Here we characterized the uptake and release of calcium in a pure melanosomal fraction obtained from frog RPE. Melanosomes take up 45Ca by a saturable system with an apparent KM of 0.5 mM. About 40% of 45Ca accumulation was insensitive to low temperature. 45Ca uptake was not affected by verapamil, nifedipine, dantrolene, vanadate, thapsigargin or cyclopiazonic acid, but it was reduced by 50% by ruthenium red, and increased by the ionophore A23187 and nigericin. Release of 45Ca-loaded was stimulated by caffeine and inositol 1,4,5 trisphosphate (IP3). Caffeine stimulated release of calcium was blocked by either ryanodine or ruthenium red, but calcium released by IP3 was not affected by heparin. No binding of 3H-IP3 was observed. The 3H-ryanodine binding sites exhibited a KB of 1.3 nM and a Bmax of 12.1 fmol/mg protein. Thus, our results suggest that melanosomes may function as intracellular organelles that regulate calcium concentration in RPE.

Sarcoplasmic reticulum in aged skeletal muscle.

A decrease in muscle mass and strength and a slowing of muscle contraction are common features of the ageing process. Recent advances in basic biochemical knowledge have provided new insights into pathogenetic mechanisms underlying age-related changes in the excitation-contraction coupling process, Ca2+-transients and isometric twitch-contraction time. Sarcoplasmic reticulum (SR) Ca2+-pumps are not basically altered in physiological ageing, but several aspects of the Ca2+-transport system remain controversial, regarding phosphorylation-dependent regulation in slow-twitch muscles, in particular. It seems that conflicting reports and divergent interpretations concerning the effect of ageing on SR Ca2+-release arise from the type of muscle, the stage of the ageing process and the animal species. A cause-effect relationship between the decrease in dihydropyridine receptors and in muscle strength is strongly suggested by studies in transgenic mice, but is unsupported by our studies with fast-twitch and slow-twitch muscles of old rats. Our experimental evidence also seems to exclude the occurrence of age-related changes in the number and in the functional behaviour of Ca2+-release channels/ryanodine receptors (RyR1), based on ¿3H-ryanodine binding studies. There is emerging, although only suggestive evidence, so far, that modulation of RyR1 by SR luminal protein calsequestrin, or the functional coupling of RyRs by FKBP-12, may be altered in ageing skeletal muscle.

Calcium-sensitivity of the SR calcium release channel in failing and nonfailing human myocardium.

Altered Ca2+ metabolism of the sarcoplasmic reticulum results in changes of the contractile behavior in failing human myocardium. The ryanodine-sensitive Ca2+ release channel of the sarcoplasmic reticulum plays a key role in the intracellular Ca2+ handling in cardiac myocytes. Recently, we showed that the density of 3H-ryanodine binding sites which correspond to the SR Ca2+ release channel in human myocardial homogenates is unchanged in failing human myocardium. However, the sensitivity of the channel towards Ca2+, which acts as the trigger signal of channel activation and thereby initiates contraction, has not yet been investigated in failing and nonfailing myocardium. Homogenates (100 micrograms protein) from hearts with dilated (DCM, n = 10) or ischemic (ICM, n = 9) cardiomyopathy were incubated with a saturating concentration of 3H-ryanodine (12 nM) in the presence of different Ca2+ concentrations ranging from 1 nM to 10 mM. For comparison, myocardium of 8 nonfailing hearts which could not be transplanted for technical reasons was investigated. Non-specific binding was determined in the presence of a high concentration (10 microM) of unlabeled ryanodine. 3H-ryanodine binding to the Ca2+ release channel showed a bell-shaped pattern with an increase in specific binding at submicromolar Ca2+ concentrations and a decrease at higher Ca2+ concentrations than 0.5 mM, whereas nonspecific binding was not influenced by different Ca2+ concentrations. In nonfailing myocardium, maximal 3H-ryanodine binding (Bmax) was 85.2 +/- 3.1 fmol/mg protein and half-maximal binding was reached at a free Ca2+ concentration of 0.25 (0.22-0.30) microM (EC50). Neither EC50 values nor maximal specific 3H-ryanodine binding differed between nonfailing and failing myocardium of both etiologies. EC50 values were 0.24 (0.23-0.26) microM (DCM, n = 10) or 0.28 (0.25-0.31) microM (ICM, n = 9), respectively. Caffeine (2 mM) and the ATP-analogon AMP-PCP (1 mM) led to a shift towards lower Ca2+ concentrations consistent with an activation of the channel by these compounds, whereas Mg2+ (0.7 mM) shifted the Ca(2+)-dependence of 3H-ryanodine binding towards higher Ca2+ concentrations indicating inhibition of channel opening. After activation of the Ca2+ release channel by caffeine or AMP-PCP as well as after the inhibition with Mg2+ EC50 values were the same in failing and nonfailing myocardium. Caffeine and AMP-PCP sensitize, whereas Mg2+ desensitizes the myocardial Ca2+ release channel to Ca2+. The determination of Ca(2+)-dependent 3H-ryanodine binding to the human myocardial Ca2+ release channel is a useful tool to investigate its open probability. Furthermore, the Ca(2+)-sensitivity and the pharmacological behavior of the human SR Ca2+ release channel are similar in failing and nonfailing myocardium.

The ryanodine binding sarcoplasmic reticulum calcium release channel in nonfailing and in failing human myocardium.

The ryanodine-sensitive Ca2+ release channel (RyaCRC) of the sarcoplasmic reticulum plays a key role in the intracellular Ca2+ handling in cardiomyocytes. Altered expression of the RyaCRC has been supposed to contribute to abnormal cellular Ca2+ handling and to myocardial dysfunction in dilated and ischemic cardiomyopathy. In the present study the 3H-ryanodine binding site in human myocardial homogenates was characterized and the density of the RyaCRC (which corresponds to the cardiac ryanodine receptor) was determined in nonfailing and in failing human myocardium. Homogenates were prepared from nonfailing left ventricular myocardium from the hearts of 5 organ donors (NF) and from failing myocardium from 14 explanted hearts of transplant recipients with end-stage heart failure resulting from dilated (DCM, n = 5) or ischemic (ICM, n = 9) cardiomyopathy. Radioligand saturation binding experiments revealed a specific, high-affinity 3H-ryanodine binding site (Kd-values: NF: 0.65 +/- 0.11 nmol/l, DCM: 0.66 +/- 0.09 nmol/l, ICM: 0.88 +/- 0.18 nmol/l; n.s.) in all preparations. Specific 3H-ryanodine binding depended on the free Ca2+ concentration in the assay. It was maximal at 3-100 micro mol/l Ca2+. The binding was inhibited by the RyaCRC antagonists ruthenium red (Ki-value: 0.32 [0.18-0.56] micromol/l, n = 5) and Mg2+ (Ki-value: 2.95 [1.23-7.11] mmol/l, n = 5). The RyaCRC density was 103.5 +/- 11.9 fmol/mg protein in nonfailing myocardium. There was no significant change in the RyaCRC density in dilated or ischemic cardiomyopathy (112.4 +/- 17.1 and 122.7 +/- 13.9 fmol/mg protein) compared to nonfailing control myocardium. In summary, 3H-ryanodine binds specifically and with high-affinity to the RyaCRC in human myocardium. There is no change in the RyaCRC density in failing myocardium of patients with DCM or ICM in comparison to non-failing controls.

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.

Decrease in myocardial ryanodine receptors and altered excitation-contraction coupling early in the development of heart failure.

Rapid ventricular pacing for 1 day reduced myocardial contractile function without inducing heart failure in conscious, chronically instrumented dogs. After 4 to 7 weeks of pacing, myocardial contractility was depressed further and overt signs of congestive heart failure, eg, ascites, dyspnea, and edema, were evident. The mechanical restitution response, a physiological index of calcium release, was depressed at 1 day of rapid ventricular pacing. Postextrasystolic potentiation was also depressed by a similar amount, 14 +/- 3%, at 1 day after pacing. The response to isoproterenol 0.2 microgram/kg per minute was depressed by a significantly greater amount (P < .05), 52 +/- 7%, at 1 day after pacing. 3H-ryanodine receptor binding fell from 1013 +/- 25 to 808 +/- 42 fmol/mg after 1 day of pacing and remained depressed at similar levels (782 +/- 61 fmol/mg) at 4 to 7 weeks when heart failure was manifest. Ryanodine receptor affinity was unchanged from control values. Neither dihydropyridine binding nor affinity for 3H-PN200-110 was changed from control levels. Within 5 days after recovery from 1 day of pacing, physiological responses to isoproterenol, postextrasystolic potentiation, and mechanical restitution recovered, as did 3H-ryanodine binding density. These findings suggest that the changes in excitation-contraction coupling and potentially the sarcoplasmic reticulum calcium release channel occur early in the development of heart failure and therefore may be important in the pathogenesis of the contractile abnormalities in this disease state.

Effect of ischemia and reperfusion on cardiac ryanodine receptors--sarcoplasmic reticulum Ca2+ channels.

We investigated the effect of ischemia and reperfusion on the cardiac ryanodine receptor, which corresponds to the sarcoplasmic reticulum Ca2+ channel. Isolated working rat hearts were subjected to 10 to 30 minutes of global ischemia, followed or not by reperfusion. Ischemia produced significant reduction in the density of high-affinity 3H-ryanodine binding sites, determined either in whole-heart homogenate (Bmax, 220 +/- 22, 203 +/- 12, and 228 +/- 14 fmol/mg protein after 10, 20, and 30 minutes of ischemia versus 298 +/- 18 fmol/mg protein in the control condition; P < .01) or in a fraction enriched in sarcoplasmic reticulum (Bmax, 1.08 +/- 0.15 pmol/mg protein after 20 minutes of ischemia versus 1.69 +/- 0.08 pmol/mg protein in the control condition; P < .01). The Kd (1.5 +/- 0.1 nmol/L) and the Ca2+ dependence of high-affinity 3H-ryanodine binding were not affected by ischemia. The density of low-affinity 3H-ryanodine binding sites was also reduced after 20 minutes of ischemia (14.0 +/- 2.3 versus 34.0 +/- 8.2 pmol/mg protein in the sarcoplasmic reticulum fraction, P < .05), without significant changes in Kd (4.7 +/- 1.2 versus 2.4 +/- 1.0 mumol/L). All these changes persisted after 20 minutes of reperfusion. Analysis of tissue fractions showed that 55% of the ryanodine binding sites were retained in the pellet of a low-speed centrifugation ("nuclear pellet") and that the effects of ischemia concerned only the receptors released in the supernatant ("postnuclear supernatant"). In parallel experiments, we evaluated the effect of ryanodine on oxalate-supported Ca2+ uptake, which represents sarcoplasmic reticulum Ca2+ uptake. As expected, we found that high concentrations of ryanodine stimulated Ca2+ uptake, owing to channel blockade. The response to 900 mumol/L ryanodine was slightly reduced in crude homogenate and significantly reduced in postnuclear supernatant obtained from ischemic hearts. In conclusion, the number of ryanodine receptors is reduced after ischemia; this effect concerns a subpopulation of the receptors, persists after reperfusion, and might contribute to modify sarcoplasmic reticulum function.

O-040 - 3H-ryanodine binding to chicken fast twitch skeletal muscle sarcoolasmic reticulum

O-040 - 3H-ryanodine binding to chicken fast twitch skeletal muscle sarcoolasmic reticulum