Mini-dystrophin expression down-regulates IP3-mediated calcium release events in resting dystrophin-deficient muscle cells

We present here evidence for the enhancement, at rest, of an inositol 1,4,5-trisphosphate (IP3)-mediated calcium signaling pathway in myotubes from dystrophin-deficient cell lines (SolC1(-)) as compared to a cell line from the same origin but transfected with mini-dystrophin (SolD(+)). With confocal microscopy, the number of sites discharging calcium (release site density [RSD]) was quantified and found more elevated in SolC1(-) than in SolD(+) myotubes. Variations of membrane potential had no significant effect on this difference, and higher resting [Ca2+]i in SolC1(-) (Marchand, E., B. Constantin, H. Balghi, M.C. Claudepierre, A. Cantereau, C. Magaud, A. Mouzou, G. Raymond, S. Braun, and C. Cognard. 2004. Exp. Cell Res. 297:363-379) cannot explain alone higher RSD. The exposure with SR Ca(2+) channel inhibitors (ryanodine and 2-APB) and phospholipase C inhibitor (U73122) significantly reduced RSD in both cell types but with a stronger effect in dystrophin-deficient SolC1(-) myotubes. Immunocytochemistry allowed us to localize ryanodine receptors (RyRs) as well as IP3 receptors (IP3Rs), IP3R-1 and IP3R-2 isoforms, indicating the presence of both RyRs-dependent and IP3-dependent release systems in both cells. We previously reported evidence for the enhancement, through a Gi protein, of the IP3-mediated calcium signaling pathway in SolC1(-) as compared to SolD(+) myotubes during a high K(+) stimulation (Balghi, H., S. Sebille, B. Constantin, S. Patri, V. Thoreau, L. Mondin, E. Mok, A. Kitzis, G. Raymond, and C. Cognard. 2006. J. Gen. Physiol. 127:171-182). Here we show that, at rest, these regulation mechanisms are also involved in the modulation of calcium release activities. The enhancement of resting release activity may participate in the calcium overload observed in dystrophin-deficient myotubes, and our findings support the hypothesis of the regulatory role of mini-dystrophin on intracellular signaling.     

Focal sarcoplasmic reticulum calcium stores and diffuse inositol 1,4,5-trisphosphate and ryanodine receptors in human myometrium

Intracellular calcium stores of human uterine myocytes in primary and second passage cell culture were visualized using the low-affinity calcium-sensitive fluorescent dye, fluo-3FF. The calcium stores appeared as numerous small (0.2-0.5 microm diameter) focal fluorescences. The stores were not depleted by exposing the cells to oxytocin or ryanodine under standard conditions. The stores were rapidly depleted by oxytocin or ryanodine exposure when sarcoplasmic reticulum (SR) calcium re-uptake was inhibited by pretreatment with thapsigargin. Immunofluorescence experiments indicated that both ryanodine and inositol 1,4,5-trisphosphate (IP(3)) receptors were smoothly distributed throughout the SR, and neither receptor co-localized with the calcium stores. Since IP(3) and ryanodine calcium channels are tightly associated with their receptor, these results suggest that SR calcium release occurs via second messenger channels that are remote from the SR calcium stores. These observations are consistent only with a mechanism for release of calcium stores where the SR serves three functions: (1) as site of calcium storage, (2) as the structure that contains the IP(3)- and ryanodine receptors and their associated release channels, and (3) as a conduit between the calcium stores and the release channels.     

Homer modulates NFAT-dependent signaling during muscle differentiation

While changes in intracellular calcium are well known to influence muscle contraction through excitation contraction coupling, little is understood of the calcium signaling events regulating gene expression through the calcineurin/NFAT pathway in muscle. Here, we demonstrate that Ca(+2) released via the inositol trisphosphate receptor (IP3R) increases nuclear entry of NFAT in undifferentiated skeletal myoblasts, but the IP3R Ca(+2) pool in differentiated myotubes promotes nuclear exit of NFAT despite a comparable quantitative change in [Ca(+2)]i. In contrast, Ca(+2) released via ryanodine receptors (RYR) increases NFAT nuclear entry in myotubes. The scaffolding protein Homer, known to interact with both IP3R and RYR, is expressed as part of the myogenic differentiation program and enhances NFAT-dependent signaling by increasing RYR Ca(+2) release. These results demonstrate that differentiated skeletal myotubes employ discrete pools of intracellular calcium to restrain (IP3R pool) or activate (RYR pool) NFAT-dependent signaling, in a manner distinct from undifferentiated myoblasts. The selective expression of Homer proteins contributes to these differentiation-dependent features of calcium signaling.     

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.     

IP3 receptor-dependent Ca2+ release modulates excitation-contraction coupling in rabbit ventricular myocytes

Inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R)-dependent Ca(2+) signaling exerts positive inotropic, but also arrhythmogenic, effects on excitation-contraction coupling (ECC) in the atrial myocardium. The role of IP(3)R-dependent sarcoplasmic reticulum (SR) Ca(2+) release in ECC in the ventricular myocardium remains controversial. Here we investigated the role of this signaling pathway during ECC in isolated rabbit ventricular myocytes. Immunoblotting of proteins from ventricular myocytes showed expression of both type 2 and type 3 IP(3)R at levels approximately 3.5-fold less than in atrial myocytes. In permeabilized myocytes, direct application of IP(3) (10 microM) produced a transient 21% increase in the frequency of Ca(2+) sparks (P < 0.05). This increase was accompanied by a 13% decrease in spark amplitude (P < 0.05) and a 7% decrease in SR Ca(2+) load (P < 0.05) and was inhibited by IP(3)R antagonists 2-aminoethoxydiphenylborate (2-APB; 20 microM) and heparin (0.5 mg/ml). In intact myocytes endothelin-1 (100 nM) was used to stimulate IP(3) production and caused a 38% (P < 0.05) increase in the amplitude of action potential-induced (0.5 Hz, field stimulation) Ca(2+) transients. This effect was abolished by the IP(3)R antagonist 2-APB (2 microM) or by using adenoviral expression of an IP(3) affinity trap that buffers cellular IP(3). Together, these data suggest that in rabbit ventricular myocytes IP(3)R-dependent Ca(2+) release has positive inotropic effects on ECC by facilitating Ca(2+) release through ryanodine receptor clusters.     

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.     

Stores not just for storage. intracellular calcium release and synaptic plasticity.

Activation of most excitatory synapses of central neurons produces calcium release signals from intracellular stores. Synaptically evoked calcium release from stores is frequently triggered by the binding of glutamate to metabotropic receptors and the subsequent activation of IP(3) receptors in spines and dendrites. There is increasing evidence for the presence of local calcium signals caused by calcium-induced calcium release (CICR) through activation of ryanodine or IP(3) receptors. Recent work on mutant mice indicates that store signaling determines activity-dependent synaptic plasticity.     

Imperatoxin a enhances Ca(2+) release in developing skeletal muscle containing ryanodine receptor type 3

Most adult mammalian skeletal muscles contain only one isoform of ryanodine receptor (RyR1), whereas neonatal muscles contain two isoforms (RyR1 and RyR3). Membrane depolarization fails to evoke calcium release in muscle cells lacking RyR1, demonstrating an essential role for this isoform in excitation-contraction coupling. In contrast, the role of RyR3 is unknown. We studied the participation of RyR3 in calcium release in wild type (containing both RyR1 and RyR3 isoforms) and RyR3-/- (containing only RyR1) myotubes in the presence or absence of imperatoxin A (IpTxa), a high-affinity agonist of ryanodine receptors. IpTxa significantly increased the amplitude and the rate of release only in wild-type myotubes. Calcium currents, recorded simultaneously with the transients, were not altered with IpTxa treatment. [(3)H]ryanodine binding to RyR1 or RyR3 was significantly increased in the presence of IpTxa. Additionally, IpTxa modified the gating and conductance level of single RyR1 or RyR3 channels when studied in lipid bilayers. Our data show that IpTxa can interact with both RyRs and that RyR3 is functional in myotubes and it can amplify the calcium release signal initiated by RyR1, perhaps through a calcium-induced mechanism. In addition, our data indicate that when RyR3-/- myotubes are voltage-clamped, the effect of IpTxa is not detected because RyR1s are under the control of the dihydropyridine receptor.     

Role for calcium in the development of ovarial patency in Heliothis virescens

Insect oocytes sequester nutritive proteins from the hemolymph under the regulation by juvenile hormone (JH), in a process called patency. Here, a pharmacological approach was used to decipher the role for calcium in ovarial patency in the moth, Heliothis virescens. Follicular epithelial cells were exposed in calcium-free or calcium-containing media to JH I, JH II or JH III alone, or in combination with various inhibitors of signal transduction. Protein kinase inhibitors, Na(+)/K(+) -ATPase inhibitor, ouabain, an inhibitor of voltage-dependent calcium channels in plasma membrane, omega-Conotoxin MVII, endoplasmic reticulum (ER) Ca(2+) -ATPase inhibitor, thapsigargin, ER inositol 1,4,5-triphosphate receptor (IP(3)R) inhibitor, 2-ABP and ER ryanodine receptor (RyR) inhibitor, ryanodine, were used. The results of our study suggest that JH II evokes patency via protein kinase C-dependent signaling pathway, and activation of Na(+)/K(+) -ATPase, similar to JH III. Response to JH II and JH III predominantly relies upon external and internal calcium stores, using voltage-dependent calcium channels, IP(3)Rs and RyRs. In contrast, regulation of patency by JH I appears to be largely calcium independent, and the calcium-dependent component of the signaling pathway likely does not use IP(3)Rs, but RyRs only. The JH II, JH III and calcium-dependent component of JH I signaling pathway probably utilize calcium/calmodulin-dependent kinase II for activation of Na(+)/K(+) -ATPase.     

Uranyl acetate modulates gene expression and protein levels of the type 2, but not type 1 inositol 1,4,5-trisphosphate receptors in mouse kidney

Nephrotoxic effect of uranium is already well documented. Nevertheless, little is known about the effect of uranium on calcium homeostasis and calcium transport systems. Calcium released from endoplasmic reticulum through special calcium release channels--inositol 1,4,5-trisphosphate receptors (IP3Rs) and ryanodine receptors (RyRs)--serves as a main source of cytosolic calcium signaling in the majority of cell types. To contribute to understanding mechanism of toxicity of the uranyl acetate (UA), we focused on modulation of the gene expression, protein levels and activity of IP3 receptor's intracellular calcium channels by UA in mouse kidney. We have found that UA did not affect mRNA and protein levels of the type 1 IP3Rs, but increased mRNA and also protein levels of the type 2 IP3 receptors in kidney. Nevertheless, IP3-induced calcium release was decreased by addition of UA. We assume that decreased activity of IP3 receptors due to the acute exposure to UA results in feedback, which triggers activation of IP3R2 expression. Thus, inhibition of calcium release and increased levels of the type 2 IP3 receptors might participate, at least partially, in UA-induced nephrotoxicity.     

Triad formation: organization and function of the sarcoplasmic reticulum calcium release channel and triadin in normal and dysgenic muscle in vitro

Excitation-contraction (E-C) coupling is thought to involve close interactions between the calcium release channel (ryanodine receptor; RyR) of the sarcoplasmic reticulum (SR) and the dihydropyridine receptor (DHPR) alpha 1 subunit in the T-tubule membrane. Triadin, a 95- kD protein isolated from heavy SR, binds both the RyR and DHPR and may thus participate in E-C coupling or in interactions responsible for the formation of SR/T-tubule junctions. Immunofluorescence labeling of normal mouse myotubes shows that the RyR and triadin co-aggregate with the DHPR in punctate clusters upon formation of functional junctions. Dysgenic myotubes with a deficiency in the alpha 1 subunit of the DHPR show reduced expression and clustering of RyR and triadin; however, both proteins are still capable of forming clusters and attaining mature cross-striated distributions. Thus, the molecular organization of the RyR and triadin in the terminal cisternae of SR as well as its association with the T-tubules are independent of interactions with the DHPR alpha 1 subunit. Analysis of calcium transients in dysgenic myotubes with fluorescent calcium indicators reveals spontaneous and caffeine-induced calcium release from intracellular stores similar to those of normal muscle; however, depolarization-induced calcium release is absent. Thus, characteristic calcium release properties of the RyR do not require interactions with the DHPR; neither do they require the normal organization of the RyR in the terminal SR cisternae. In hybrids of dysgenic myotubes fused with normal cells, both action potential- induced calcium transients and the normal clustered organization of the RyR are restored in regions expressing the DHPR alpha 1 subunit.     

Local production of O2- by NAD(P)H oxidase in the sarcoplasmic reticulum of coronary arterial myocytes: cADPR-mediated Ca2+ regulation

The present study was designed to determine whether the sarcoplasmic reticulum (SR) could locally produce superoxide (O2-) via NAD(P)H oxidase (NOX) in coronary arterial myocytes (CAMs) and to address whether cADPR-RyR/Ca2+ signaling pathway regulates this local O2- production from the SR. Using confocal microscopic imaging analysis in intact single CAMs, a cell-permeable indicator CM-H2DCFDA for dynamic changes in intracellular ROS (in green color) and a highly selective ER-Tracker Red dye for tracking of the SR were found co-localized. A quantitative analysis based on the intensity of different spectra demonstrated a local O2- production derived from the SR. M(1)-receptor agonist, oxotremorine (Oxo) and a Ca2+ ionophore, A23187, time-dependently increased this O2- production colocalized with the SR. NOX inhibitors, diphenylene iodonium (DPI) and apocynin (Apo), or superoxide dismutase (SOD) and catalase, and Nox4 (a major intracellular NOX subunit) siRNA all substantially blocked this local production of O2-, demonstrating an involvement of NOX. This SR-derived O2- production was also abolished by the inhibitors of cyclic ADP-ribose (cADPR)-mediated Ca2+ signaling, such as nicotinamide (Nicot, 6 mM), ryanodine (Rya, 50 muM) or 8-Br-cADPR (30 microM). However, IP3 antagonist, 2-APB (50 microM) had no effect. In CAMs transfected with siRNA of ADP-ribosyl cyclase or RyR, this SR O2- production was attenuated. Electron spin resonance (ESR) spectromic assay in purified SR also demonstrated the production of O2- that was dependent on NOX activity and Ca2+ concentrations. These results provide direct evidence that O2- could be locally produced via NOX on the SR and that this local O2- producing system is controlled by cADPR-RyR/Ca2+ signaling pathway.     

Protease-activated receptor-1-induced calcium signaling in gingival fibroblasts is mediated by sarcoplasmic reticulum calcium release and extracellular calcium influx

Thrombin is a serine protease activated during injury and inflammation. Thrombin and other proteases generated by periodontal pathogens affect the behavior of periodontal cells via activation of protease-activated receptors (PARs). We noted that thrombin and PAR-1 agonist peptide stimulated intracellular calcium levels ([Ca2+]i) of gingival fibroblasts (GF). This increase of [Ca2+]i was inhibited by EGTA and verapamil. U73122 and neomycin inhibited thrombin- and PAR-1-induced [Ca2+]i. Furthermore, 2-APB (75-100 microM, inositol triphosphate [IP3] receptor antagonist), thapsigargin (1 microM), SKF-96365 (200 microM) and W7 (50 and 100 microM) also suppressed the PAR-1- and thrombin-induced [Ca2+]i. However, H7 (100, 200 microM) and ryanodine showed little effects. Blocking Ca2+ efflux from mitochondria by CGP37157 (50, 100 microM) inhibited both thrombin- and PAR-1-induced [Ca2+]i. Thrombin induced the IP3 production of GF within 30-seconds of exposure, which was inhibited by U73122. These results indicate that mitochondrial calcium efflux and calcium-calmodulin pathways are related to thrombin and PAR-1 induced [Ca2+]i in GF. Thrombin-induced [Ca2+]i of GF is mainly due to PAR-1 activation, extracellular calcium influx via L-type calcium channel, PLC activation, then IP3 binding to IP3 receptor in sarcoplasmic reticulum, which leads to intracellular calcium release and subsequently alters cell membrane capacitative calcium entry.     

Extracellular calcium participates in responses to acetylcholine in Xenopus oocytes

We tested the contribution of extracellular calcium (Ca2+) to membrane electrical responses to acetylcholine (ACh) in native Xenopus oocytes. Removal of Cao caused a decrease in both the rapid (D1) and the slow (D2) chloride currents that comprise the common depolarizing response to ACh in native oocyte. The effect of Ca2+o removal on the muscarinic response was mimicked by the addition of 1 mM Mn2+, an effective antagonist of calcium influx, though not by antagonists of voltage-sensitive calcium channels. When oocytes were challenged with ACh in Ca2(+)-free medium, subsequent addition of 1.8 mM CaCl2 resulted in a rapid, often transient, depolarizing current. Similarly to the Ca2+o-dependent component of membrane electrical responses, the Ca2(+)-evoked current was reversibly abolished by Mn2+, though not by antigonists of voltage-sensitive calcium channels. Depletion of cellular calcium potentiated the Ca2(+)-evoked current, implying negative feedback of calcium channels by calcium. Injection of 10-100 fmol of inositol 1,4,5-trisphosphate (IP3) resulted in a two-component depolarizing current. IP3 injection promoted the appearance of Ca2+o-evoked current that was significantly potentiated by previous calcium depletion. We suggest that activation of cell-membrane muscarinic receptors causes opening of apparently voltage-insensitive and verapamil or diltiazem-resistant calcium channels. These channels may be activated by IP3 or its metabolites, which increase following the activation of cell membrane receptors coupled to a phospholipase C. The channels may be identical to receptor-operated channels described in other model systems.     

Possible link of different slow calcium signals generated by membrane potential and hormones to differential gene expression in cultured muscle cells

The study of fluorescent calcium signals from cultured rat myotubes has provided interesting results in the past few years. Both K+ depolarization and tetanic electrical stimulation were shown to produce slow Ca2+ signals, unrelated to contraction and associated to regulation of gene expression in cultured rat myotubes. We studied the effect of IGF-I, insulin and testosterone on intracellular Ca2+ in cultured muscle cells. Insulin produced a fast (< 1 s) and transient [Ca2+] increase lasting less than 10 s. IGF-I induced a transient [Ca2+] increase, reaching a fluorescence peak 6 s after stimulus, to return to basal values after 60 s. Testosterone induced delayed (35 s) and long lasting (100-200 s) signals, frequently associated with oscillations. IGF-I, testosterone and electrical stimulation-induced Ca2+ signals were shown to be dependent on IP3 production. All of these Ca2+ signals were blocked by inhibitors of the IP3 pathway. On the other hand, insulin-induced Ca2+ increase was dependent on ryanodine receptors and blocked by either nifedipine or ryanodine. The different intracellular Ca2+ patterns produced by electrical stimulation, testosterone, IGF-I and insulin, may help to understand the role of intracellular calcium kinetics in the regulation of gene expression by various stimuli in skeletal muscle cells.