Nitric oxide signals postovulatory aging-induced abortive spontaneous egg activation in rats

Abstract

Objective

The aim of this study was to determine whether an increase of intracellular nitric oxide (NO) level signals postovulatory aging-induced abortive spontaneous egg activation (SEA) in rats.

Methods

Freshly ovulated eggs (arrested at metaphase-II stage; M-II) were cultured in vitro for 3 hours to induce postovulatory egg aging. The morphological changes, inducible nitric oxide synthase (iNOS) expression, NO, cytosolic free Ca²⁺, 3′,5′ cyclic guanosine monophosphate (cGMP), cell division cycle 25B (Cdc25B) and Wee1 levels, specific phosphorylation (pThr-14/Tyr-15) as well as total cyclin-dependent kinases-1 (Cdk1) (PSTAIRE) levels were analyzed.

Results

Postovulatory aging induced generation of NO possibly through an iNOS-mediated pathway. The increase in NO level was associated with augmented cytosolic free Ca²⁺ as well as cGMP levels in aged eggs. A significant increase in Wee1 level and decrease of Cdc25B level were observed in aged eggs. An accumulation of phosphorylated Cdk1 (pThr-14/Tyr-15) level was observed in aged eggs, while total Cdk1 (PSTAIR) level remained unchanged.

Conclusion

Our study demonstrates that generation of NO through an iNOS-mediated pathway increases cytosolic free Ca2+and cGMP levels. High levels of these signal molecules trigger the accumulation of phosphorylated Cdk1 in aged eggs. Thus, NO signals the accumulation of phosphorylated Cdk1 and induces postovulatory aging-induced abortive SEA in the rat.     

Morphological,cellular and molecular changes during postovulatory egg aging in mammals

Postovulatory aging is associated with several morphological, cellular and molecular changes that deteriorate egg quality either by inducing abortive spontaneous egg activation (SEA) or by egg apoptosis. The reduced egg quality results in poor fertilization rate, embryo quality and reproductive outcome. Although postovulatory aging-induced abortive SEA has been reported in several mammalian species, the molecular mechanism(s) underlying this process remains to be elucidated. The postovulatory aging-induced morphological and cellular changes are characterized by partial cortical granules exocytosis, zona pellucida hardening, exit from metaphase-II (M-II)arrest and initiation of extrusion of second polar body in aged eggs. The molecular changes include reduction of adenosine 3'',5''- cyclic monophosphate (cAMP) level, increase of reactive oxygen species (ROS) and thereby cytosolic free calcium (Ca²⁺) level. Increased levels of cAMP and/or ROS trigger accumulation of Thr-14/Tyr-15 phosphorylated cyclin-dependent kinase 1 (Cdk1) on one hand and degradation of cyclin B1 through ubiquitin-mediated proteolysis on the other hand to destabilize maturation promoting factor (MPF). The destabilized MPF triggers postovulatory aging-induced abortive SEA and limits various assisted reproductive technologies (ARTs) outcome in several mammalian species. Use of certain drugs that can either increase cAMP or reduce ROS level would prevent postovulatory aging-induced deterioration in egg quality so that more number of good quality eggs can be made available to improve ART outcome in mammals including human.     

Maturation promoting factor destabilization facilitates postovulatory aging‐mediated abortive spontaneous egg activation in rat

The present study was designed to investigate whether destabilization of maturation promoting factor (MPF) leads to postovulatory aging‐mediated abortive spontaneous egg activation (SEA). If so, we wished to determine whether changes in Wee‐1 as well as Emi2 levels are associated with MPF destabilization during postovulatory aging‐mediated abortive SEA in rats eggs aged in vivo. For this purpose, sexually immature female rats were given a single injection (20 IU IM) of pregnant mare serum gonadotropin for 48 h followed by single injection of human chorionic gonadotropin (20 IU). Ovulated eggs were collected after 14, 17, 19 and 21 h post‐hCG surge to induce postovulatory aging in vivo. The morphological changes, Wee1, phosphorylation status of cyclin dependent kinase 1(Cdk1), early mitotic inhibitor 2 (Emi2), anaphase promoting complex/cyclosome (APC/C), cyclin B1, mitotic arrest deficient protein (MAD2) levels and Cdk1 activity were analyzed. The increased Wee 1 level triggered phosphorylation of Thr‐14/Tyr‐15 and dephosphorylation of Thr‐161 residues of Cdk1. The decrease of Emi2 level was associated with increased APC/C level and decreased cyclin B1 level. Changes in phosphorylation status of Cdk1 and reduced cyclin B1 level resulted in destabilization of MPF. The destabilized MPF finally led to postovulatory aging‐mediated abortive SEA in rat eggs. It was concluded that the increase of Wee 1 but decrease of Emi2 level triggers MPF destabilization and thereby postovulatory aging‐mediated abortive SEA in rat eggs.     

Intracellular Zinc Modulates Cardiac Ryanodine Receptor-mediated Calcium Release

Aberrant Zn²⁺ homeostasis is a hallmark of certain cardiomyopathies associated with altered contractile force. In this study, we addressed whether Zn²⁺ modulates cardiac ryanodine receptor gating and Ca²⁺ dynamics in isolated cardiomyocytes. We reveal that Zn²⁺ is a high affinity regulator of RyR2 displaying three modes of operation. Picomolar free Zn²⁺ concentrations potentiate RyR2 responses, but channel activation is still dependent on the presence of cytosolic Ca²⁺. At concentrations of free Zn²⁺ >1 nm, Zn²⁺ is the main activating ligand, and the dependence on Ca²⁺ is removed. Zn²⁺ is therefore a higher affinity activator of RyR2 than Ca²⁺. Millimolar levels of free Zn²⁺ were found to inhibit channel openings. In cardiomyocytes, consistent with our single channel results, we show that Zn²⁺ modulates both the frequency and amplitude of Ca²⁺ waves in a concentration-dependent manner and that physiological levels of Zn²⁺ elicit Ca²⁺ release in the absence of activating levels of cytosolic Ca²⁺. This highlights a new role for intracellular Zn²⁺ in shaping Ca²⁺ dynamics in cardiomyocytes through modulation of RyR2 gating.     

Mechanism of calsequestrin regulation of single cardiac ryanodine receptor in normal and pathological conditions

Release of Ca²⁺ from the sarcoplasmic reticulum (SR) drives contractile function of cardiac myocytes. Luminal Ca²⁺ regulation of SR Ca²⁺ release is fundamental not only in physiology but also in physiopathology because abnormal luminal Ca²⁺ regulation is known to lead to arrhythmias, catecholaminergic polymorphic ventricular tachycardia (CPVT), and/or sudden cardiac arrest, as inferred from animal model studies. Luminal Ca²⁺ regulates ryanodine receptor (RyR)2-mediated SR Ca²⁺ release through mechanisms localized inside the SR; one of these involves luminal Ca²⁺ interacting with calsequestrin (CASQ), triadin, and/or junctin to regulate RyR2 function.CASQ2-RyR2 regulation was examined at the single RyR2 channel level. Single RyR2s were incorporated into planar lipid bilayers by the fusion of native SR vesicles isolated from either wild-type (WT), CASQ2 knockout (KO), or R33Q-CASQ2 knock-in (KI) mice. KO and KI mice have CPVT-like phenotypes. We show that CASQ2(WT) action on RyR2 function (either activation or inhibition) was strongly influenced by the presence of cytosolic MgATP. Function of the reconstituted CASQ2(WT)–RyR2 complex was unaffected by changes in luminal free [Ca²⁺] (from 0.1 to 1 mM). The inhibition exerted by CASQ2(WT) association with the RyR2 determined a reduction in cytosolic Ca²⁺ activation sensitivity. RyR2s from KO mice were significantly more sensitive to cytosolic Ca²⁺ activation and had significantly longer mean open times than RyR2s from WT mice. Sensitivity of RyR2s from KI mice was in between that of RyR2 channels from KO and WT mice. Enhanced cytosolic RyR2 Ca²⁺ sensitivity and longer RyR2 open times likely explain the CPVT-like phenotype of both KO and KI mice.     

Rise of intracellular Ca2+ level causes the decrease of cyclin B1 and Mos in the newt eggs at fertilization

Unfertilized eggs of the newt, Cynops pyrrhogaster, are arrested at the second meiotic metaphase, with activity of the M‐phase promoting factor (MPF) maintained at a high level. After fertilization, the eggs resume the cell cycle, and emit the second polar body. When the change in [Ca²⁺]_i in the fertilized eggs was monitored by aequorin, an early increase in [Ca²⁺]_i was observed 5–10 min after insemination and continued for about 30 sec. A late increase in [Ca²⁺]_i then occurred 10–15 min after fertilization and continued for 30–40 min. The injection of 1,2‐Bis (2 aminophenoxy) ethane‐N,N,N′,N′,‐tetraacetic acid (BAPTA) into unfertilized eggs inhibited reinitiation of the cell cycle after fertilization. Western blot analysis with antibodies against cyclin B1 or Mos indicated that both cyclin B1 and Mos were present in unfertilized eggs, but both disappeared within 30 min after fertilization. Treatment with Ca²⁺‐ionophore decreased both cyclin B1 and Mos. Chymotryptic activity in Cynops egg extracts was not significantly increased after fertilization or activation by treatment with the Ca²⁺‐ionophore. No change in [Ca²⁺]_i was observed following treatment with cycloheximide, but the amount of both cyclin B1 and Mos rapidly decreased. These results indicate that resumption of meiosis in Cynops eggs is induced by an increase in [Ca²⁺]_i at fertilization, which causes degradation of both cyclin B1 and Mos by inhibition of de novo synthesis of those proteins. Mol. Reprod. Dev. 53:341–349, 1999. © 1999 Wiley‐Liss, Inc.     

Ca<Superscript>2+</Superscript>-Dependent Phosphorylation of RyR2 Can Uncouple Channel Gating from Direct Cytosolic Ca<Superscript>2+</Superscript> Regulation

Phosphorylation of the cardiac ryanodine receptor (RyR2) is thought to be important not only for normal cardiac excitation-contraction coupling but also in exacerbating abnormalities in Ca²⁺ homeostasis in heart failure. Linking phosphorylation to specific changes in the single-channel function of RyR2 has proved very difficult, yielding much controversy within the field. We therefore investigated the mechanistic changes that take place at the single-channel level after phosphorylating RyR2 and, in particular, the idea that PKA-dependent phosphorylation increases RyR2 sensitivity to cytosolic Ca²⁺. We show that hyperphosphorylation by exogenous PKA increases open probability (P _o) but, crucially, RyR2 becomes uncoupled from the influence of cytosolic Ca²⁺; lowering [Ca²⁺] to subactivating levels no longer closes the channels. Phosphatase (PP1) treatment reverses these gating changes, returning the channels to a Ca²⁺-sensitive mode of gating. We additionally found that cytosolic incubation with Mg²⁺/ATP in the absence of exogenously added kinase could phosphorylate RyR2 in approximately 50% of channels, thereby indicating that an endogenous kinase incorporates into the bilayer together with RyR2. Channels activated by the endogenous kinase exhibited identical changes in gating behavior to those activated by exogenous PKA, including uncoupling from the influence of cytosolic Ca²⁺. We show that the endogenous kinase is both Ca²⁺-dependent and sensitive to inhibitors of PKC. Moreover, the Ca²⁺-dependent, endogenous kinase–induced changes in RyR2 gating do not appear to be related to phosphorylation of serine-2809. Further work is required to investigate the identity and physiological role of this Ca²⁺-dependent endogenous kinase that can uncouple RyR2 gating from direct cytosolic Ca²⁺ regulation.     

High cytosolic free calcium level signals apoptosis through mitochondria-caspase mediated pathway in rat eggs cultured in vitro

The present study was aimed to find out whether an increase of cytosolic free calcium level induces egg apoptosis through mitochondria-caspase mediated pathway. To increase cytosolic free calcium level and morphological apoptotic changes, ovulated eggs were cultured in Ca²⁺/Mg²⁺ free media-199 with or without various concentrations of calcium ionophore (0.5, 1, 2, 3, 4 μM) for 3 h in vitro. The morphological apoptotic changes, cytosolic free calcium level, hydrogen peroxide (H₂O₂) concentration, catalase activity, cytochrome c concentration, caspase-9 and caspase-3 activities and DNA fragmentation were analyzed. Calcium ionophore induced morphological apoptotic features in a concentration-dependent manner followed by degeneration at higher concentrations (3 and 4 μM). Calcium ionophore increased cytosolic free calcium level, induced generation of hydrogen peroxide (H₂O₂) and inhibited catalase activity in treated eggs. The increased H₂O₂ concentration was associated with increased cytochrome c concentration, caspase-9 and caspase-3 activities that resulted in the induction of morphological features characteristic of egg apoptosis. The increased caspase-3 activity finally induced DNA fragmentation as evidenced by TUNEL positive staining in calcium ionophore-treated eggs. These findings suggest that high cytosolic free calcium level induces generation of H₂O₂ that leads to egg apoptosis through mitochondria-caspase mediated pathway.     

The EF-hand Ca2+ Binding Domain Is Not Required for Cytosolic Ca2+ Activation of the Cardiac Ryanodine Receptor

Activation of the cardiac ryanodine receptor (RyR2) by elevating cytosolic Ca²⁺ is a central step in the process of Ca²⁺-induced Ca²⁺ release, but the molecular basis of RyR2 activation by cytosolic Ca²⁺ is poorly defined. It has been proposed recently that the putative Ca²⁺ binding domain encompassing a pair of EF-hand motifs (EF1 and EF2) in the skeletal muscle ryanodine receptor (RyR1) functions as a Ca²⁺ sensor that regulates the gating of RyR1. Although the role of the EF-hand domain in RyR1 function has been studied extensively, little is known about the functional significance of the corresponding EF-hand domain in RyR2. Here we investigate the effect of mutations in the EF-hand motifs on the Ca²⁺ activation of RyR2. We found that mutations in the EF-hand motifs or deletion of the entire EF-hand domain did not affect the Ca²⁺-dependent activation of [³H]ryanodine binding or the cytosolic Ca²⁺ activation of RyR2. On the other hand, deletion of the EF-hand domain markedly suppressed the luminal Ca²⁺ activation of RyR2 and spontaneous Ca²⁺ release in HEK293 cells during store Ca²⁺ overload or store overload-induced Ca²⁺ release (SOICR). Furthermore, mutations in the EF2 motif, but not EF1 motif, of RyR2 raised the threshold for SOICR termination, whereas deletion of the EF-hand domain of RyR2 increased both the activation and termination thresholds for SOICR. These results indicate that, although the EF-hand domain is not required for RyR2 activation by cytosolic Ca²⁺, it plays an important role in luminal Ca²⁺ activation and SOICR.     

Single Ryanodine Receptor Channel Basis of Caffeine's Action on Ca Sparks

Caffeine (1, 3, 7-trimethylxanthine) is a widely used pharmacological agonist of the cardiac ryanodine receptor (RyR2) Ca²⁺ release channel. It is also a well-known stimulant that can produce adverse side effects, including arrhythmias. Here, the action of caffeine on single RyR2 channels in bilayers and Ca²⁺ sparks in permeabilized ventricular cardiomyocytes is defined. Single RyR2 caffeine activation depended on the free Ca²⁺ level on both sides of the channel. Cytosolic Ca²⁺ enhanced RyR2 caffeine affinity, whereas luminal Ca²⁺ essentially scaled maximal caffeine activation. Caffeine activated single RyR2 channels in diastolic quasi-cell-like solutions (cytosolic MgATP, pCa 7) with an EC₅₀ of 9.0 ± 0.4 mM. Low-dose caffeine (0.15 mM) increased Ca²⁺ spark frequency ∼75% and single RyR2 opening frequency ∼150%. This implies that not all spontaneous RyR2 openings during diastole are associated with Ca²⁺ sparks. Assuming that only the longest openings evoke sparks, our data suggest that a spark may result only when a spontaneous single RyR2 opening lasts >6 ms.     

The H29D Mutation Does Not Enhance Cytosolic Ca2+ Activation of the Cardiac Ryanodine Receptor

The N-terminal domain of the cardiac ryanodine receptor (RyR2) harbors a large number of naturally occurring mutations that are associated with stress-induced ventricular tachyarrhythmia and sudden death. Nearly all these disease-associated N-terminal mutations are located at domain interfaces or buried within domains. Mutations at these locations would alter domain-domain interactions or the stability/folding of domains. Recently, a novel RyR2 mutation H29D associated with ventricular arrhythmia at rest was found to enhance the activation of single RyR2 channels by diastolic levels of cytosolic Ca²⁺. Unlike other N-terminal disease-associated mutations, the H29D mutation is located on the surface of the N-terminal domain. It is unclear how this surface-exposed H29D mutation that does not appear to interact with other parts of the RyR2 structure could alter the intrinsic properties of the channel. Here we carried out detailed functional characterization of the RyR2-H29D mutant at the molecular and cellular levels. We found that the H29D mutation has no effect on the basal level or the Ca²⁺ dependent activation of [³H]ryanodine binding to RyR2, the cytosolic Ca²⁺ activation of single RyR2 channels, or the cytosolic Ca²⁺- or caffeine-induced Ca²⁺ release in HEK293 cells. In addition, the H29D mutation does not alter the propensity for spontaneous Ca²⁺ release or the thresholds for Ca²⁺ release activation or termination. Furthermore, the H29D mutation does not have significant impact on the thermal stability of the N-terminal region (residues 1–547) of RyR2. Collectively, our data show that the H29D mutation exerts little or no effect on the function of RyR2 or on the folding stability of the N-terminal region. Thus, our results provide no evidence that the H29D mutation enhances the cytosolic Ca²⁺ activation of RyR2.     

Calcium pool size modulates the sensitivity of the ryanodine receptor channel and calcium-dependent ATPase of heavy sarcoplasmic reticulum to extravesicular free calcium concentration

We have examined calcium cycling and associated ATP consumption by isolated heavy sarcoplasmic reticulum (HSR) vesicles incubated in conditions believed to exist in resting muscle. Our goals were to estimate the magnitude of calcium cycling under those conditions and identify the main mechanisms involved in its regulation. The integrity of the HSR vesicles was documented by the retention of [¹⁴C]-sucrose and electron microscopy. HSR actively exchanged Ca²⁺ with the medium through a partially open ryanodine-binding channel (RyR), as evidenced by the rapid attainment of a steady-state gradient between HSR and medium, which was promptly increased by the closure of the channel with ruthenium red (RR) or collapsed by its opening with caffeine. The ATP dependency was evidenced by the sustained ATP consumption after the steady state was attained and by the abrogation of the gradient following inhibition of the pump with thapsigargin (Tg) or the omission of ATP. When HSR vesicles were incubated in a comparatively large pool of calcium (≈1 μmol/mg HSR protein), ATP consumption was 1–1.5 μmol × [min × mg protein]⁻¹ at 0.1 μM free Ca²⁺. Under such conditions, the main regulator of the sarcoplasmic Ca²⁺-dependent ATPase (SERCA) was extravesicular-free Ca²⁺ concentration, with a four- to fivefold increase between 0.1 and 2 μM Ca²⁺, whereas RyR channel activity and the replenishment of the HSR vesicles had only a modest effect on ATP consumption. When calcium pool size was reduced to 0.1 μmol/mg HSR protein, a steady state was established at a lower level of HSR calcium. In spite of a slightly lower free extravesicular Ca²⁺ at equilibrium (≈0.07 μM following an initial concentration of 0.1 μM), both ATP consumption and the open probability of the RyR channel were increased by a factor of three to five. Compared to the large calcium pool, the sensitivity of both RyR channel and SERCA to extravesicular free Ca²⁺ concentration as well as to caffeine and RR was markedly enhanced. Conclusions: (1) In conditions present in resting muscle, HSR calcium is in dynamic equilibrium with the medium through a partially open RyR channel, which requires continuous ATP hydrolysis. (2) The availability of calcium is a major determinant of the sensitivity of both RyR channel and SERCA to free extravesicular Ca²⁺ and possibly other stimuli. (3) These observations are consistent with the concept that calcium cycling in resting muscle may account for a significant fraction of muscle energy demands and further suggest that restricting calcium availability may enhance the energetic demands of this process. J. Cell. Physiol. 175:283–294, 1998. © 1998 Wiley-Liss, Inc.     

Regulation of sarcoplasmic reticulum Ca2+ release by cytosolic glutathione in rabbit ventricular myocytes

Of the major cellular antioxidant defenses, glutathione (GSH) is particularly important in maintaining the cytosolic redox potential. Whereas the healthy myocardium is maintained at a highly reduced redox state, it has been proposed that oxidation of GSH can affect the dynamics of Ca²⁺-induced Ca²⁺ release. In this study, we used multiple approaches to define the effects of oxidized glutathione (GSSG) on ryanodine receptor (RyR)-mediated Ca²⁺ release in rabbit ventricular myocytes. To investigate the role of GSSG on sarcoplasmic reticulum (SR) Ca²⁺ release induced by the action potential, we used the thiol-specific oxidant diamide to increase intracellular GSSG in intact myocytes. To more directly assess the effect of GSSG on RyR activity, we introduced GSSG within the cytosol of permeabilized myocytes. RyR-mediated Ca²⁺ release from the SR was significantly enhanced in the presence of GSSG. This resulted in decreased steady-state diastolic [Ca²⁺]_SR, increased SR Ca²⁺ fractional release, and increased spark- and non-spark-mediated SR Ca²⁺ leak. Single-channel recordings from RyR’s incorporated into lipid bilayers revealed that GSSG significantly increased RyR activity. Moreover, oxidation of RyR in the form of intersubunit crosslinking was present in intact myocytes treated with diamide and permeabilized myocytes treated with GSSG. Blocking RyR crosslinking with the alkylating agent N-ethylmaleimide prevented depletion of SR Ca²⁺ load induced by diamide. These findings suggest that elevated cytosolic GSSG enhances SR Ca²⁺ leak due to redox-dependent intersubunit RyR crosslinking. This effect can contribute to abnormal SR Ca²⁺ handling during periods of oxidative stress.     

A calcium-induced calcium release mechanism mediated by calsequestrin

Calcium (Ca²⁺)-induced Ca²⁺ release (CICR) is widely accepted as the principal mechanism linking electrical excitation and mechanical contraction in cardiac cells. The CICR mechanism has been understood mainly based on binding of cytosolic Ca²⁺ with ryanodine receptors (RyRs) and inducing Ca²⁺ release from the sarcoplasmic reticulum (SR). However, recent experiments suggest that SR lumenal Ca²⁺ may also participate in regulating RyR gating through calsequestrin (CSQ), the SR lumenal Ca²⁺ buffer. We investigate how SR Ca²⁺ release via RyR is regulated by Ca²⁺ and calsequestrin (CSQ). First, a mathematical model of RyR kinetics is derived based on experimental evidence. We assume that the RyR has three binding sites, two cytosolic sites for Ca²⁺ activation and inactivation, and one SR lumenal site for CSQ binding. The open probability (P_o) of the RyR is found by simulation under controlled cytosolic and SR lumenal Ca²⁺. Both peak and steady-state P_o effectively increase as SR lumenal Ca²⁺ increases. Second, we incorporate the RyR model into a CICR model that has both a diadic space and the junctional SR (jSR). At low jSR Ca²⁺ loads, CSQs are more likely to bind with the RyR and act to inhibit jSR Ca²⁺ release, while at high SR loads CSQs are more likely to detach from the RyR, thereby increasing jSR Ca²⁺ release. Furthermore, this CICR model produces a nonlinear relationship between fractional jSR Ca²⁺ release and jSR load. These findings agree with experimental observations in lipid bilayers and cardiac myocytes.     

?-Blocker Timolol Prevents Arrhythmogenic Ca2+ Release and Normalizes Ca2+ and Zn2+ Dyshomeostasis in Hyperglycemic Rat Heart

Defective cardiac mechanical activity in diabetes results from alterations in intracellular Ca²⁺ handling, in part, due to increased oxidative stress. Beta-blockers demonstrate marked beneficial effects in heart dysfunction with scavenging free radicals and/or acting as an antioxidant. The aim of this study was to address how β-blocker timolol-treatment of diabetic rats exerts cardioprotection. Timolol-treatment (12-week), one-week following diabetes induction, prevented diabetes-induced depressed left ventricular basal contractile activity, prolonged cellular electrical activity, and attenuated the increase in isolated-cardiomyocyte size without hyperglycemic effect. Both in vivo and in vitro timolol-treatment of diabetic cardiomyocytes prevented the altered kinetic parameters of Ca²⁺ transients and reduced Ca²⁺ loading of sarcoplasmic reticulum (SR), basal intracellular free Ca²⁺ and Zn²⁺ ([Ca²⁺]_i and [Zn²⁺]_i), and spatio-temporal properties of the Ca²⁺ sparks, significantly. Timolol also antagonized hyperphosphorylation of cardiac ryanodine receptor (RyR2), and significantly restored depleted protein levels of both RyR2 and calstabin2. Western blot analysis demonstrated that timolol-treatment also significantly normalized depressed levels of some [Ca²⁺]_i-handling regulators, such as Na⁺/Ca²⁺ exchanger (NCX) and phospho-phospholamban (pPLN) to PLN ratio. Incubation of diabetic cardiomyocytes with 4-mM glutathione exerted similar beneficial effects on RyR2-macromolecular complex and basal levels of both [Ca²⁺]_i and [Zn²⁺]_i, increased intracellular Zn²⁺ hyperphosphorylated RyR2 in a concentration-dependent manner. Timolol also led to a balanced oxidant/antioxidant level in both heart and circulation and prevented altered cellular redox state of the heart. We thus report, for the first time, that the preventing effect of timolol, directly targeting heart, seems to be associated with a normalization of macromolecular complex of RyR2 and some Ca²⁺ handling regulators, and prevention of Ca²⁺ leak, and thereby normalization of both [Ca²⁺]_i and [Zn²⁺]_i homeostasis in diabetic rat heart, at least in part by controlling the cellular redox status of hyperglycemic cardiomyocytes.     

Identification of Changes in the Functional Profile of the Cardiac Ryanodine Receptor Caused by the Coupled Gating Phenomenon

The objective of this work was to identify and further characterize potential changes in the functional profile of the cardiac ryanodine receptor (RyR2) channel caused by the coupled gating phenomenon. By reconstituting an ion channel into a planar lipid membrane, we showed that coupled RyR2 channels were activated by cytosolic Ca²⁺ with similar efficacy and potency as reported for the single RyR2 channel. In contrast, all examined parameters of gating kinetics were affected by the functional interaction between channels. Ignoring brief closings during main open events, the average open and closed times were considerably prolonged and the frequency of opening was reduced. Interestingly, when luminal Ca²⁺ was used as a charge carrier, Ca²⁺-activated coupled RyR2 channels did not exhibit a sudden switch from slow to fast gating kinetics at an open probability of 0.5 as reported for the single RyR2 channel. Regarding flicker gating, the average closed time was significantly shorter and the frequency of closing was greatly enhanced. Furthermore, in contrast to the single RyR2 channel, both parameters for coupled channels were independent of cytosolic Ca²⁺. Selected permeation properties of coupled RyR2 channels were comparable to those found for the single RyR2 channel. The Ca²⁺ current amplitude-luminal Ca²⁺ relationship displayed a simple saturation and the channel selectivity for Ba²⁺ and Ca²⁺ ions was similar. Our results suggest that the major targets influenced by coupled gating are likely the gates of individual RyR2 channels recruited into a functional complex, thus ensuring the correlation of Ca²⁺ fluxes.     

The role of the plasma-membrane Ca2+-ATPase in Ca2+ homeostasis in Sinapis alba root hairs

The regulation of cytosolic Ca²⁺ has been investigated in growing root-hair cells of Sinapis alba L. with special emphasis on the role of the plasmamembrane Ca²⁺-ATPase. For this purpose, erythrosin B was used to inhibit the Ca²⁺-ATPase, and the Ca²⁺ ionophore A₂₃₁₈₇ was applied to manipulate cytosolic free [Ca²⁺] which was then measured with Ca²⁺-selective microelectrodes. (i) At 0.01 M, A₂₃₁₈₇ had no effect on the membrane potential but enhanced the Ca²⁺ permeability of the plasma membrane. Higher concentrations of this ionophore strongly depolarized the cells, also in the presence of cyanide. (ii) Unexpectedly, A₂₃₁₈₇ first caused a decrease in cytosolic Ca²⁺ by 0.2 to 0.3 pCa units and a cytosolic acidification by about 0.5 pH units, (iii) The depletion of cytosolic free Ca²⁺ spontaneously reversed and became an increase, a process which strongly depended on the external Ca²⁺ concentration, (iv) Upon removal of A₂₃₁₈₇, the cytosolic free [Ca²⁺] returned to its steady-state level, a process which was inhibited by erythrosin B. We suggest that the first reaction to the intruding Ca²⁺ is an activation of Ca²⁺ transporters (e.g. ATPases at the endoplasmic reticulum and the plasma membrane) which rapidly remove Ca²⁺ from the cytosol. The two observations that after the addition of A₂₃₁₈₇, (i) Ca²⁺ gradients as steep as-600 mV could be maintained and (ii) the cytosolic pH rapidly and immediately decreased without recovery indicate that the Ca²⁺-exporting plasma-membrane ATPase is physiologically connected to the electrochemical pH gradient, and probably works as an nH⁺/Ca²⁺-ATPase. Based on the finding that the Ca²⁺-ATPase inhibitor erythrosin B had no effect on cytosolic Ca²⁺, but caused a strong Ca²⁺ increase after the addion of A₂₃₁₈₇ we conclude that these cells, at least in the short term, have enough metabolic energy to balance the loss in transport activity caused by inhibition of the primary Ca²⁺-pump. We further conclude that this ATPase is a major Ca²⁺ regulator in stress situations where the cytosolic Ca²⁺ has been shifted from its steady-state level, as may be the case during processes of signal transduction.Abbreviations and Symbols EB erythrosin B - E_m membrane potential - pCa negative logarithm of the Ca²⁺ concentration This work was supported by the Deutche Forschungsgemeinschaft (H.F.) and the Alexander-von-Humboldt-Foundation (A.T.).     

Caffeine-induced Release of Intracellular Ca2+ from Chinese Hamster Ovary Cells Expressing Skeletal Muscle Ryanodine Receptor : Effects on Full-Length and Carboxyl-Terminal Portion of Ca2+Release Channels

The ryanodine receptor (RyR)/Ca²⁺ release channel is an essential component of excitation–contraction coupling in striated muscle cells. To study the function and regulation of the Ca²⁺ release channel, we tested the effect of caffeine on the full-length and carboxyl-terminal portion of skeletal muscle RyR expressed in a Chinese hamster ovary (CHO) cell line. Caffeine induced openings of the full length RyR channels in a concentration-dependent manner, but it had no effect on the carboxyl-terminal RyR channels. CHO cells expressing the carboxyl-terminal RyR proteins displayed spontaneous changes of intracellular [Ca²⁺]. Unlike the native RyR channels in muscle cells, which display localized Ca²⁺ release events (i.e., “Ca²⁺ sparks” in cardiac muscle and “local release events” in skeletal muscle), CHO cells expressing the full length RyR proteins did not exhibit detectable spontaneous or caffeine-induced local Ca²⁺ release events. Our data suggest that the binding site for caffeine is likely to reside within the amino-terminal portion of RyR, and the localized Ca²⁺ release events observed in muscle cells may involve gating of a group of Ca²⁺ release channels and/or interaction of RyR with muscle-specific proteins.     

Homeostatic and stimulus-induced coupling of the L-type Ca2+ channel to the ryanodine receptor in the hippocampal neuron in slices

Activity-dependent increase in cytosolic calcium ([Ca²⁺]_i) is a prerequisite for many neuronal functions. We previously reported a strong direct depolarization, independent of glutamate receptors, effectively caused a release of Ca²⁺ from ryanodine-sensitive stores and induced the synthesis of endogenous cannabinoids (eCBs) and eCB-mediated responses. However, the cellular mechanism that initiated the depolarization-induced Ca²⁺-release is not completely understood. In the present study, we optically recorded [Ca²⁺]_i from CA1 pyramidal neurons in the hippocampal slice and directly monitored miniature Ca²⁺ activities and depolarization-induced Ca²⁺ signals in order to determine the source(s) and properties of [Ca²⁺]_i-dynamics that could lead to a release of Ca²⁺ from the ryanodine receptor. In the absence of depolarizing stimuli, spontaneously occurring miniature Ca²⁺ events were detected from a group of hippocampal neurons. This miniature Ca²⁺ event persisted in the nominal Ca²⁺-containing artificial cerebrospinal fluid (ACSF), and increased in frequency in response to the bath-application of caffeine and KCl. In contrast, nimodipine, the antagonist of the L-type Ca²⁺ channel (LTCC), a high concentration of ryanodine, the antagonist of the ryanodine receptor (RyR), and thapsigargin (TG) reduced the occurrence of the miniature Ca²⁺ events. When a brief puff-application of KCl was given locally to the soma of individual neurons in the presence of glutamate receptor antagonists, these neurons generated a transient increase in the [Ca²⁺]_i in the dendrosomal region. This [Ca²⁺]_i-transient was sensitive to nimodipine, TG, and ryanodine suggesting that the [Ca²⁺]_i-transient was caused primarily by the LTCC-mediated Ca²⁺-influx and a release of Ca²⁺ from RyR. We observed little contribution from N- or P/Q-type Ca²⁺ channels. The coupling between LTCC and RyR was direct and independent of synaptic activities. Immunohistochemical study revealed a cellular localization of LTCC and RyR in a juxtaposed configuration in the proximal dendrites and soma. We conclude in the hippocampal CA1 neuron that: (1) homeostatic fluctuation of the resting membrane potential may be sufficient to initiate functional coupling between LTCC and RyR; (2) the juxtaposed localization of LTCC and RyR has anatomical advantage of synchronizing a Ca²⁺-release from RyR upon the opening of LTCC; and (3) the synchronized Ca²⁺-release from RyR occurs immediately after the activation of LTCC and determines the peak amplitude of depolarization-induced global increase in dendrosomal [Ca²⁺]_i.