Nitroxyl triggers Ca2+ release from skeletal and cardiac sarcoplasmic reticulum by oxidizing ryanodine receptors

The biological activity of nitric oxide (NO) and NO-donors has been extensively investigated yet few studies have examined those of nitroxyl (HNO) species even though both exist in chemical equilibrium but oxidize thiols by different reaction mechanisms: S-nitrosation versus disulfide bond formation. Here, sodium trioxodinitrate (Na2N2O3; Angeli's salt; ANGS) was used as an HNO donor to investigate its effects on skeletal (RyR1) and cardiac (RyR2) ryanodine receptors. At steady-state concentrations of nanomoles/L, HNO induced a rapid Ca2+ release from sarcoplasmic reticulum (SR) vesicles then the reducing agent dithiothreitol (DTT) reversed the oxidation by HNO resulting in Ca2+ re-uptake by SR vesicles. With RyR1 channel proteins reconstituted in planar bilayers, HNO added to the cis-side increased the open probability (Po) from 0.056+/-0.026 to 0.270+/-0.102 (P<0.005, n=4) then DTT (3 mM) reduced Po to 0.096+/-0.040 (P<0.01, n=4). In parallel experiments, the time course of HNO production from ANGS was monitored by EPR and UV spectroscopy and compared with the rate of SR Ca2+ release indicating that picomolar concentrations of HNO triggered SR Ca2+ release. Controls showed that the hydroxyl radical scavenger, phenol did not alter ANGS-induced SR Ca2+ release, indicating that hydroxyl radical production from ANGS did not account for Ca2+ release from the SR. The findings indicate that HNO is a more potent activator of RyR1 than NO and that HNO activation of RyRs may contribute to NO's activation of RyRs and to the therapeutic effects of HNO-releasing prodrugs in heart failure.     

Molecular regulation of cardiac ryanodine receptor ion channel

The release of Ca(2+) ions from intracellular stores is a key step in a wide variety of cellular functions. In striated muscle, the release of Ca(2+) from the sarcoplasmic reticulum (SR) leads to muscle contraction. Ca(2+) release occurs through large, high-conductance Ca(2+) release channels, also known as ryanodine receptors (RyRs) because they bind the plant alkaloid ryanodine with high affinity and specificity. The RyRs are isolated as 30S protein complexes comprised of four 560 kDa RyR2 subunits and four 12 kDa FK506 binding protein (FKBP12) subunits. Multiple endogenous effector molecules and posttranslational modifications regulate the RyRs. This review focuses on current research toward understanding the control of the isolated cardiac Ca(2+) release channel/ryanodine receptor (RyR2) by Ca(2+), calmodulin, thiol oxidation/reduction and nitrosylation, and protein phosphorylation.     

Sub-plasmalemmal [Ca2+]i upstroke in myocytes of the guinea-pig small intestine evoked by muscarinic stimulation: IP3R-mediated Ca2+ release induced by voltage-gated Ca2+ entry

Membrane depolarization triggers Ca(2+) release from the sarcoplasmic reticulum (SR) in skeletal muscles via direct interaction between the voltage-gated L-type Ca(2+) channels (the dihydropyridine receptors; VGCCs) and ryanodine receptors (RyRs), while in cardiac muscles Ca(2+) entry through VGCCs triggers RyR-mediated Ca(2+) release via a Ca(2+)-induced Ca(2+) release (CICR) mechanism. Here we demonstrate that in phasic smooth muscle of the guinea-pig small intestine, excitation evoked by muscarinic receptor activation triggers an abrupt Ca(2+) release from sub-plasmalemmal (sub-PM) SR elements enriched with inositol 1,4,5-trisphosphate receptors (IP(3)Rs) and poor in RyRs. This was followed by a lesser rise, or oscillations in [Ca(2+)](i). The initial abrupt sub-PM [Ca(2+)](i) upstroke was all but abolished by block of VGCCs (by 5 microM nicardipine), depletion of intracellular Ca(2+) stores (with 10 microM cyclopiazonic acid) or inhibition of IP(3)Rs (by 2 microM xestospongin C or 30 microM 2-APB), but was not affected by block of RyRs (by 50-100 microM tetracaine or 100 microM ryanodine). Inhibition of either IP(3)Rs or RyRs attenuated phasic muscarinic contraction by 73%. Thus, in contrast to cardiac muscles, excitation-contraction coupling in this phasic visceral smooth muscle occurs by Ca(2+) entry through VGCCs which evokes an initial IP(3)R-mediated Ca(2+) release activated via a CICR mechanism.     

Luminal Ca2+-regulated Mg2+ inhibition of skeletal RyRs reconstituted as isolated channels or coupled clusters

In resting muscle, cytoplasmic Mg(2+) is a potent inhibitor of Ca(2+) release from the sarcoplasmic reticulum (SR). It is thought to inhibit calcium release channels (RyRs) by binding both to low affinity, low specificity sites (I-sites) and to high affinity Ca(2+) sites (A-sites) thus preventing Ca(2+) activation. We investigate the effects of luminal and cytoplasmic Ca(2+) on Mg(2+) inhibition at the A-sites of skeletal RyRs (RyR1) in lipid bilayers, in the presence of ATP or modified by ryanodine or DIDS. Mg(2+) inhibits RyRs at the A-site in the absence of Ca(2+), indicating that Mg(2+) is an antagonist and does not simply prevent Ca(2+) activation. Cytoplasmic Ca(2+) and Cs(+) decreased Mg(2+) affinity by a competitive mechanism. We describe a novel mechanism for luminal Ca(2+) regulation of Ca(2+) release whereby increasing luminal [Ca(2+)] decreases the A-site affinity for cytoplasmic Mg(2+) by a noncompetitive, allosteric mechanism that is independent of Ca(2+) flow. Ryanodine increases the Ca(2+) sensitivity of the A-sites by 10-fold, which is insufficient to explain the level of activation seen in ryanodine-modified RyRs at nM Ca(2+), indicating that ryanodine activates independently of Ca(2+). We describe a model for ion binding at the A-sites that predicts that modulation of Mg(2+) inhibition by luminal Ca(2+) is a significant regulator of Ca(2+) release from the SR. We detected coupled gating of RyRs due to luminal Ca(2+) permeating one channel and activating neighboring channels. This indicated that the RyRs existed in stable close-packed rafts within the bilayer. We found that luminal Ca(2+) and cytoplasmic Mg(2+) did not compete at the A-sites of single open RyRs but did compete during multiple channel openings in rafts. Also, luminal Ca(2+) was a stronger activator of multiple openings than single openings. Thus it appears that RyRs are effectively "immune" to Ca(2+) emanating from their own pore but sensitive to Ca(2+) from neighboring channels.     

Oxidation and reduction of pig skeletal muscle ryanodine receptors

Time-dependent effects of cysteine modification were compared in skeletal ryanodine receptors (RyRs) from normal pigs and RyR(MH) (Arg(615) to Cys(615)) from pigs susceptible to malignant hyperthermia, using the oxidizing reagents 4,4'-dithiodipyridine (4, 4'-DTDP) and 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB) or the reducing agent dithiothreitol (DTT). Normal and RyR(MH) channels responded similarly to all reagents. DTNB (1 mM), either cytoplasmic (cis) or luminal (trans), or 1 mM 4,4'-DTDP (cis) activated RyRs, introducing an additional long open time constant. 4,4'-DTDP (cis), but not DTNB, inhibited channels after >5 min. Activation and inhibition were relieved by DTT (1-10 mM). DTT (10 mM, cytoplasmic or luminal), without oxidants, activated RyRs, and activation reversed with 1 mM DTNB. Control RyR activity was maintained with 1 mM DTNB and 10 mM DTT present on the same or opposite sides of the bilayer. We suggest that 1) 4,4'-DTDP and DTNB covalently modify RyRs by oxidizing activating or inhibiting thiol groups; 2) a modified thiol depresses mammalian skeletal RyR activity under control conditions; 3) both the activating thiols and the modified thiols, accessible from either cytoplasm or lumen, reside in the transmembrane region; 4) some cardiac sulfhydryls are unavailable in skeletal RyRs; and 5) Cys(615) in RyR(MH) is functionally unimportant in redox cycling.     

Calcium mobilization is required for peroxynitrite-mediated enhancement of spontaneous transient outward currents in arteriolar smooth muscle cells

Transiently local release of Ca(2+) from the sarcoplasmic reticulum (SR) activates nearby Ca(2+)-activated K(+) channels to produce spontaneous transient outward currents (STOCs) in smooth muscle cells. The purpose of the present study was to investigate the possible effect of peroxynitrite (ONOO(-)) on STOCs in mesenteric arteriolar smooth muscle cells (ASMCs) and decide whether Ca(2+) mobilization was involved in STOCs alteration by ONOO(-). STOCs were recorded and characterized using the perforated whole-cell patch-clamp configuration. The results demonstrated that STOCs activity was greatly suppressed by removal of extracellular Ca(2+); by addition of nifedipine, a specific inhibitor of L-type voltage-gated Ca(2+) channels (VGCCs); or by addition of ryanodine, a SR ryanodine receptors (RyRs) blocker. In contrast, both caffeine, a RyR activator, and 2-aminoethoxydiphenylborate (2-APB), a membrane-permeable inositol 1,4,5-trisphosphate receptors, (IP3R) antagonist, increased STOCs activity. 3-morpholinosydnonimine (SIN-1), an ONOO(-) donor, at concentrations of 20-200 microM, induced a dose-dependent enhancement of STOCs in ASMCs and led to conspicuous increases in STOCs frequency and amplitude, which were prevented by prior exposure to low external Ca(2+) (200 nM), ryanodine (10 microM), or nifedipine (10 microM). In contrast, caffeine (0.5 mM) did not further stimulate STOCs in ASMCs preincubated with SIN-1, and pretreatment with 2-APB (50 microM) had little effect on ONOO(-) -induced STOCs activation. These findings suggest that complex Ca(2+)-mobilizing pathways, including external Ca2+ influx through VGCCs activation and subsequent internal Ca(2+) release through RyRs but not IP3Rs, are involved in ONOO(-)mediated STOCs enhancement in ASMCs.     

Shape, size, and distribution of Ca(2+) release units and couplons in skeletal and cardiac muscles.

Excitation contraction (e-c) coupling in skeletal and cardiac muscles involves an interaction between specialized junctional domains of the sarcoplasmic reticulum (SR) and of exterior membranes (either surface membrane or transverse (T) tubules). This interaction occurs at special structures named calcium release units (CRUs). CRUs contain two proteins essential to e-c coupling: dihydropyridine receptors (DHPRs), L-type Ca(2+) channels of exterior membranes; and ryanodine receptors (RyRs), the Ca(2+) release channels of the SR. Special CRUs in cardiac muscle are constituted by SR domains bearing RyRs that are not associated with exterior membranes (the corbular and extended junctional SR or EjSR). Functional groupings of RyRs and DHPRs within calcium release units have been named couplons, and the term is also loosely applied to the EjSR of cardiac muscle. Knowledge of the structure, geometry, and disposition of couplons is essential to understand the mechanism of Ca(2+) release during muscle activation. This paper presents a compilation of quantitative data on couplons in a variety of skeletal and cardiac muscles, which is useful in modeling calcium release events, both macroscopic and microscopic ("sparks").     

Hypochlorous acid modifies calcium release channel function from skeletal muscle sarcoplasmic reticulum.

We have previously demonstrated that H2O2 at millimolar concentrations induces Ca(2+) release from actively loaded sarcoplasmic reticulum (SR) vesicles and induces biphasic [(3)H]ryanodine binding behavior. Considering that hypochlorous acid (HOCl) is a related free radical and has been demonstrated to be a more effective oxidant of proteins, we evaluated the effects of HOCl on sarcoplasmic reticulum Ca(2+)-channel release mechanism. In a concentration-dependent manner, HOCl activates the SR Ca(2+) release channel and induces rapid release of Ca from actively loaded vesicles. HOCl-induced Ca(2+) release is inhibited in the presence of millimolar concentrations of DMSO. High-affinity [(3)H]ryanodine binding is also enhanced at concentrations from 10 to 100 microM. At HOCl concentrations of >100 microM, equilibrium binding is inhibited. HOCl stimulation of binding is inhibited by the addition of dithiothreitol. The direct interaction between HOCl and the Ca(2+) release mechanism was further demonstrated in single-channel reconstitution experiments. HOCl, at 20 microM, activated the Ca(2+) release channel after fusion of a SR vesicle to a bilayer lipid membrane. At 40 microM, Ca(2+)-channel activity was inhibited. Pretreatment of SR vesicles with HOCl inhibited the fluorescence development of a fluorogenic probe specific to thiol groups critical to channel function. These results suggest that HOCl at micromolar concentrations can modify SR Ca(2+) handling.     

Inhibition of Ca(2+) sparks by ruthenium red in permeabilized rat ventricular myocytes

We have compared the effects of the sarcoplasmic reticulum (SR) Ca(2+) release inhibitor, ruthenium red (RR), on single ryanodine receptor (RyR) channels in lipid bilayers, and on Ca(2+) sparks in permeabilized rat ventricular myocytes. Ruthenium red at 5 microM inhibited the open probability (P(o)) of RyRs approximately 20-50-fold, without significantly affecting the conductance or mean open time of the channel. At the same concentration, RR inhibited the frequency of Ca(2+) sparks in permeabilized myocytes by approximately 10-fold, and reduced the amplitude of large amplitude events (with most probable localization on the line scan) by approximately 3-fold. According to our theoretical simulations, performed with a numerical model of Ca(2+) spark formation, this reduction in Ca(2+) spark amplitude corresponds to an approximately 4-fold decrease in Ca(2+) release flux underlying Ca(2+) sparks. Ruthenium red (5 microM) increased the SR Ca(2+) content by approximately 2-fold (from 151 to 312 micromol/l cytosol). Considering the degree of inhibition of local Ca(2+) release events, the increase in SR Ca(2+) load by RR, and the lack of effects of RR on single RyR open time and conductance, we have estimated that Ca(2+) sparks under normal conditions are generated by openings of at least 10 single RyRs.     

An integrative model of the cardiac ventricular myocyte incorporating local control of Ca2+ release

The local control theory of excitation-contraction (EC) coupling in cardiac muscle asserts that L-type Ca(2+) current tightly controls Ca(2+) release from the sarcoplasmic reticulum (SR) via local interaction of closely apposed L-type Ca(2+) channels (LCCs) and ryanodine receptors (RyRs). These local interactions give rise to smoothly graded Ca(2+)-induced Ca(2+) release (CICR), which exhibits high gain. In this study we present a biophysically detailed model of the normal canine ventricular myocyte that conforms to local control theory. The model formulation incorporates details of microscopic EC coupling properties in the form of Ca(2+) release units (CaRUs) in which individual sarcolemmal LCCs interact in a stochastic manner with nearby RyRs in localized regions where junctional SR membrane and transverse-tubular membrane are in close proximity. The CaRUs are embedded within and interact with the global systems of the myocyte describing ionic and membrane pump/exchanger currents, SR Ca(2+) uptake, and time-varying cytosolic ion concentrations to form a model of the cardiac action potential (AP). The model can reproduce both the detailed properties of EC coupling, such as variable gain and graded SR Ca(2+) release, and whole-cell phenomena, such as modulation of AP duration by SR Ca(2+) release. Simulations indicate that the local control paradigm predicts stable APs when the L-type Ca(2+) current is adjusted in accord with the balance between voltage- and Ca(2+)-dependent inactivation processes as measured experimentally, a scenario where common pool models become unstable. The local control myocyte model provides a means for studying the interrelationship between microscopic and macroscopic behaviors in a manner that would not be possible in experiments.     

Role of FKBP12.6 in hypoxia- and norepinephrine-induced Ca2+ release and contraction in pulmonary artery myocytes

The cellular and molecular processes underlying the regulation of ryanodine receptor (RyR) Ca(2+) release in smooth muscle cells (SMCs) are incompletely understood. Here we show that FKBP12.6 proteins are expressed in pulmonary artery (PA) smooth muscle and associated with type-2 RyRs (RyR2), but not RyR1, RyR3, or IP(3) receptors (IP(3)Rs) in PA sarcoplasmic reticulum. Application of FK506, which binds to FKBPs and dissociates these proteins from RyRs, induced an increase in [Ca(2+)](i) and Ca(2+)-activated Cl(-) and K(+) currents in freshly isolated PASMCs, whereas cyclosporin, an agent known to inhibit calcineurin but not to interact with FKBPs, failed to induce an increase in [Ca(2+)](i). FK506-induced [Ca(2+)](i) increase was completely blocked by the RyR antagonist ruthenium red and ryanodine, but not the IP(3)R antagonist heparin. Hypoxic Ca(2+) response and hypoxic vasoconstriction were significantly enhanced in FKBP12.6 knockout mouse PASMCs. FK506 or rapamycin pretreatment also enhanced hypoxic increase [Ca(2+)](i), but did not alter caffeine-induced Ca(2+) release (SR Ca(2+) content) in PASMCs. Norepinephrine-induced Ca(2+) release and force generation were also markedly enhanced in PASMCs from FKBP12.6 null mice. These findings suggest that FKBP12.6 plays an important role in hypoxia- and neurotransmitter-induced Ca(2+) and contractile responses by regulating the activity of RyRs in PASMCs.     

Role of FKBP12.6 in cADPR-induced activation of reconstituted ryanodine receptors from arterial smooth muscle

cADP ribose (cADPR) serves as second messenger to activate the ryanodine receptors (RyRs) of the sarcoplasmic reticulum (SR) and mobilize intracellular Ca(2+) in vascular smooth muscle cells. However, the mechanisms mediating the effect of cADPR remain unknown. The present study was designed to determine whether FK-506 binding protein 12.6 (FKBP12.6), an accessory protein of the RyRs, plays a role in cADPR-induced activation of the RyRs. A 12.6-kDa protein was detected in bovine coronary arterial smooth muscle (BCASM) and cultured CASM cells by being immunoblotted with an antibody against FKBP12, which also reacted with FKBP12.6. With the use of planar lipid bilayer clamping techniques, FK-506 (0.01-10 microM) significantly increased the open probability (NP(O)) of reconstituted RyR/Ca(2+) release channels from the SR of CASM. This FK-506-induced activation of RyR/Ca(2+) release channels was abolished by pretreatment with anti-FKBP12 antibody. The RyRs activator cADPR (0.1-10 microM) markedly increased the activity of RyR/Ca(2+) release channels. In the presence of FK-506, cADPR did not further increase the NP(O) of RyR/Ca(2+) release channels. Addition of anti-FKBP12 antibody also completely blocked cADPR-induced activation of these channels, and removal of FKBP12.6 by preincubation with FK-506 and subsequent gradient centrifugation abolished cADPR-induced increase in the NP(O) of RyR/Ca(2+) release channels. We conclude that FKBP12.6 plays a critical role in mediating cADPR-induced activation of RyR/Ca(2+) release channels from the SR of BCASM.     

Detection of reversible protein thiol modifications in tissues

Oxidation/reduction reactions of protein thiol groups (PSH) have been implicated in many physiological and pathological processes. Although many new techniques for separation and identification of modified cysteinyl residues in proteins have been developed, critical assessment of reagents and sample processing often are overlooked. We carefully compared the effectiveness of N-ethylmaleimide (NEM), iodoacetamide (IAM), and iodoacetic acid (IAA) in alkylating protein thiols and found that NEM required less reagent (125 vs. 1000 mol:mol excess), required less time (4 min vs. 4h), and was more effective at lower pHs (4.3 vs. 8.0) in comparison with IAM and IAA. The relative efficacy of dithiothreitol (DTT) and tris(2-carboxyethyl)phosphine (TCEP) for reducing protein disulfides suspended in NaPO(4) buffer or MeOH was assessed, and no differences in total normalized fluorescence were detected at the concentrations tested (10-100mM); however, individual band resolution appeared better in samples reduced with DTT in MeOH. In addition, we found that oxidation ex vivo was minimized in tissue samples that were homogenized in aqueous buffers containing excess molar quantities of NEM compared with samples homogenized in MeOH containing NEM. Using NEM for thiol alkylation, DTT for disulfide reduction, and mBBr for labeling the reduced disulfide and fluorimetric detection, we were able to generate an in-gel standard curve and quantitate total disulfide contents within biological samples as well as to identify changes in specific protein bands by scanning densitometry. We demonstrated that reagents and techniques we have identified for disulfide detection in complex samples are also applicable to two-dimensional electrophoresis separations.     

Phosphatidylinositol 3,5-bisphosphate increases intracellular free Ca2+ in arterial smooth muscle cells and elicits vasocontraction

Phosphoinositide (3,5)-bisphosphate [PI(3,5)P(2)] is a newly identified phosphoinositide that modulates intracellular Ca(2+) by activating ryanodine receptors (RyRs). Since the contractile state of arterial smooth muscle depends on the concentration of intracellular Ca(2+), we hypothesized that by mobilizing sarcoplasmic reticulum (SR) Ca(2+) stores PI(3,5)P(2) would increase intracellular Ca(2+) in arterial smooth muscle cells and cause vasocontraction. Using immunohistochemistry, we found that PI(3,5)P(2) was present in the mouse aorta and that exogenously applied PI(3,5)P(2) readily entered aortic smooth muscle cells. In isolated aortic smooth muscle cells, exogenous PI(3,5)P(2) elevated intracellular Ca(2+), and it also contracted aortic rings. Both the rise in intracellular Ca(2+) and the contraction caused by PI(3,5)P(2) were prevented by antagonizing RyRs, while the majority of the PI(3,5)P(2) response was intact after blockade of inositol (1,4,5)-trisphosphate receptors. Depletion of SR Ca(2+) stores with thapsigargin or caffeine and/or ryanodine blunted the Ca(2+) response and greatly attenuated the contraction elicited by PI(3,5)P(2). The removal of extracellular Ca(2+) or addition of verapamil to inhibit voltage-dependent Ca(2+) channels reduced but did not eliminate the Ca(2+) or contractile responses to PI(3,5)P(2). We also found that PI(3,5)P(2) depolarized aortic smooth muscle cells and that LaCl(3) inhibited those aspects of the PI(3,5)P(2) response attributable to extracellular Ca(2+). Thus, full and sustained aortic contractions to PI(3,5)P(2) required the release of SR Ca(2+), probably via the activation of RyR, and also extracellular Ca(2+) entry via voltage-dependent Ca(2+) channels.     

Termination of cardiac Ca(2+) sparks: an investigative mathematical model of calcium-induced calcium release

A Ca(2+) spark arises when a cluster of sarcoplasmic reticulum (SR) channels (ryanodine receptors or RyRs) opens to release calcium in a locally regenerative manner. Normally triggered by Ca(2+) influx across the sarcolemmal or transverse tubule membrane neighboring the cluster, the Ca(2+) spark has been shown to be the elementary Ca(2+) signaling event of excitation-contraction coupling in heart muscle. However, the question of how the Ca(2+) spark terminates remains a central, unresolved issue. Here we present a new model, "sticky cluster," of SR Ca(2+) release that simulates Ca(2+) spark behavior and enables robust Ca(2+) spark termination. Two newly documented features of RyR behavior have been incorporated in this otherwise simple model: "coupled gating" and an opening rate that depends on SR lumenal [Ca(2+)]. Using a Monte Carlo method, local Ca(2+)-induced Ca(2+) release from clusters containing between 10 and 100 RyRs is modeled. After release is triggered, Ca(2+) flux from RyRs diffuses into the cytosol and binds to intracellular buffers and the fluorescent Ca(2+) indicator fluo-3 to produce the model Ca(2+) spark. Ca(2+) sparks generated by the sticky cluster model resemble those observed experimentally, and Ca(2+) spark duration and amplitude are largely insensitive to the number of RyRs in a cluster. As expected from heart cell investigation, the spontaneous Ca(2+) spark rate in the model increases with elevated cytosolic or SR lumenal [Ca(2+)]. Furthermore, reduction of RyR coupling leads to prolonged model Ca(2+) sparks just as treatment with FK506 lengthens Ca(2+) sparks in heart cells. This new model of Ca(2+) spark behavior provides a "proof of principle" test of a new hypothesis for Ca(2+) spark termination and reproduces critical features of Ca(2+) sparks observed experimentally.     

Different roles of ryanodine receptors and inositol (1,4,5)-trisphosphate receptors in adrenergically stimulated contractions of small arteries

The functions of ryanodine receptors (RyRs) and inositol (1,4,5)-trisphosphate receptors [Ins(1,4,5)P(3)Rs] in adrenergically activated contractions of pressurized rat mesenteric small arteries were investigated. Caffeine (20 mM) but not phenylephrine (PE; 10 microM) facilitated the depletion of smooth muscle sarcoplasmic reticulum (SR) Ca(2+) stores by ryanodine (40 microM). In ryanodine-treated SR-depleted arteries, 1) Ca(2+) sparks were absent, 2) low concentrations of PE failed to elicit either vasoconstriction or normal asynchronous propagating Ca(2+) waves, and 3) high [PE] induced abnormally slow oscillatory contractions (vasomotion) and synchronous Ca(2+) oscillations. In ryanodine-treated SR-depleted arteries denuded of endothelium, high [PE] induced steady contraction and steady elevation of intracellular [Ca(2+)]. In contrast, 2-aminoethyl diphenylborate (2-APB), a putative blocker of Ins(1,4,5)P(3)Rs, produced opposite effects to ryanodine: 1) Ca(2+) sparks were present; 2) Ca(2+) waves were absent; 3) caffeine-releasable Ca(2+) stores were intact; and 4) PE, even at high concentrations on endothelial-denuded arteries, failed to elicit contraction, asynchronous Ca(2+) waves, or synchronous Ca(2+) oscillations or maintained elevated [Ca(2+)]. We conclude that 1) Ins(1,4,5)P(3)Rs are essential for adrenergically induced asynchronous Ca(2+) waves and the associated steady vasoconstriction, 2) RyRs are not appreciably opened during adrenergic activation (because PE did not facilitate the development of the effects of ryanodine), and 3) Ins(1,4,5)P(3)Rs are not essential for Ca(2+) sparks. This provides an explanation of the fact that adrenergic stimulation decreases the frequency of Ca(2+) sparks (previously reported) while simultaneously increasing the frequency of asynchronous propagating Ca(2+) waves; different SR Ca(2+)-release channels are involved.     

Arg(615)Cys substitution in pig skeletal ryanodine receptors increases activation of single channels by a segment of the skeletal DHPR II-III loop

The effect of peptides, corresponding to sequences in the skeletal muscle dihydropyridine receptor II-III loop, on Ca(2+) release from sarcoplasmic reticulum (SR) and on ryanodine receptor (RyR) calcium release channels have been compared in preparations from normal and malignant hyperthermia (MH)-susceptible pigs. Peptide A (Thr(671)-Leu(690); 36 microM) enhanced the rate of Ca(2+) release from normal SR (SR(N)) and from SR of MH-susceptible muscle (SR(MH)) by 10 +/- 3.2 nmole/mg/min and 76 +/- 9.7 nmole/mg/min, respectively. Ca (2+) release from SR(N) or SR(MH) was not increased by control peptide NB (Gly(689)-Lys(708)). AS (scrambled A sequence; 36 microM) did not alter Ca (2+) release from SR(N), but increased release from SR(MH) by 29 +/- 4.9 nmoles/mg/min. RyR channels from MH-susceptible muscle (RyR(MH)) were up to about fourfold more strongly activated by peptide A (> or =1 nM) than normal RyR channels (RyR(N)) at -40 mV. Neither NB or AS activated RyR(N). RyR(MH) showed an approximately 1.8-fold increase in mean current with 30 microM AS. Inhibition at +40 mV was stronger in RyR(MH) and seen with peptide A (> or = 0.6 microM) and AS (> or = 0.6 microM), but not NB. These results show that the Arg(615)Cys substitution in RyR(MH) has multiple effects on RyRs. We speculate that enhanced DHPR activation of RyRs may contribute to increased Ca(2+) release from SR in MH-susceptible muscle.     

Activation and inhibition of purified skeletal muscle calcium release channel by NO donors in single channel current recordings.

The actions of the nitric oxide (NO) donors 1-hydroxy-2-oxo-3-(N-methyl-3-aminopropyl)-3 methyl-1-triazine (NOC-7), S-nitrosoacetylcysteine (CySNO) and S-nitrosoglutathione (GSNO) on the purified calcium release channel (ryanodine receptor) of rabbit skeletal muscle were determined by single channel current recordings. In addition, the activation of the NO donor modulated calcium release channel by the sulfhydryl oxidizing organic mercurial compound 4-(chloromercuri)phenylsulfonic acid (4-CMPS) was investigated. NOC-7 (0.1 and 0.3 mM) and CySNO (0.4 and 0.8 mM) increased the open probability (P(o)) of the calcium release channel at activating calcium concentrations (20-100 microM Ca(2+)) by 60-100%, with no effect on the current amplitude; this activation was abolished by the specific sulfhydryl reducing agent DTT. High concentrations of CySNO (1.6-2 mM) decreased P(o). Activation by GSNO (1 mM) was observed in two thirds of the experiments, but 2 mM and 4 mM GSNO markedly reduced P(o) at activating Ca(2+) (20-100 microM). In contrast to 4-CMPS, NOC-7 or GSNO had no effect at subactivating free Ca(2+) (0.6 microM). 4-CMPS further increased the open probability of NOC-7- or CySNO-stimulated channels and reversed transiently the reduced open probability of CySNO or GSNO inhibited channels at activating free Ca(2+). High concentrations of GSNO did not prevent channel activation of 4-CMPS at subactivating free Ca(2+). The NOC-7-, CySNO- or GSNO-modified channels were completely blocked by ruthenium red. It is suggested that nitrosylation/oxidation of sulfhydryls by NO donors and oxidation of sulfhydryls by 4-CMPS affect different cysteine residues essential in the gating of the calcium release channel.     

Ryanodine receptor function in newborn rat heart

The role of ryanodine receptor (RyR) in cardiac excitation-contraction (E-C) coupling in newborns (NB) is not completely understood. To determine whether RyR functional properties change during development, we evaluated cellular distribution and functionality of sarcoplasmic reticulum (SR) in NB rats. Sarcomeric arrangement of immunostained SR Ca(2+)-ATPase (SERCA2a) and the presence of sizeable caffeine-induced Ca2+ transients demonstrated that functional SR exists in NB. E-C coupling properties were then defined in NB and compared with those in adult rats (AD). Ca2+ transients in NB reflected predominantly sarcolemmal Ca2+ entry, whereas the RyR-mediated component was approximately 13%. Finally, the RyR density and functional properties at the single-channel level in NB were compared with those in AD. Ligand binding assays revealed that in NB, RyR density can be up to 36% of that found in AD, suggesting that some RyRs do not contribute to the Ca2+ transient. To test the hypothesis that RyR functional properties change during development, we incorporated single RyRs into lipid bilayers. Our results show that permeation and gating kinetics of NB RyRs are identical to those of AD. Also, endogenous ligands had similar effects on NB and AD RyRs: sigmoidal Ca2+ dependence, stronger Mg(2+)-induced inhibition at low cytoplasmic Ca2+ concentrations, comparable ATP-activating potency, and caffeine sensitivity. These observations indicate that NB rat heart contains fully functional RyRs and that the smaller contribution of RyR-mediated Ca2+ release to the intracellular Ca2+ transient in NB is not due to different single RyR channel properties or to the absence of functional intracellular Ca2+ stores.