Scielo RSS <![CDATA[Biological Research]]> https://scielo.conicyt.cl/rss.php?pid=0716-976020040004&lang=en vol. 37 num. 4 lang. en <![CDATA[SciELO Logo]]> https://scielo.conicyt.cl/img/en/fbpelogp.gif https://scielo.conicyt.cl https://scielo.conicyt.cl/scielo.php?script=sci_arttext&pid=S0716-97602004000400001&lng=en&nrm=iso&tlng=en <![CDATA[<b>Calcium signaling</b>: <b>A historical account</b>]]> https://scielo.conicyt.cl/scielo.php?script=sci_arttext&pid=S0716-97602004000400002&lng=en&nrm=iso&tlng=en <![CDATA[<b>Functional implications of RyR-DHPR relationships in skeletal and cardiac muscles </b>]]> https://scielo.conicyt.cl/scielo.php?script=sci_arttext&pid=S0716-97602004000400003&lng=en&nrm=iso&tlng=en Dihydropyridine receptors (DHPRs) and ryanodine receptors (RyRs) interact during EC coupling within calcium release units, CRUs. The location of the two channels and their positioning are related to their role in EC coupling. als DHPR and RyR1 of skeletal muscle form interlocked arrays. Groups of four DHPRs (forming a tetrad) are located on alternate RyR1s. This association provides the structural framework for reciprocal signaling between the two channels. RyR3 are present in some skeletal muscles in association with RyR1 and in ratios up to 1:1. RyR3 neither induce formation of tetrads by DHPRs nor sustain EC coupling. RyR3 are located in a parajunctional position, in proximity of the RyR1-DHPR complexes, and they may be indirectly activated by calcium liberated via the RyR1 channels. RyR2 have two locations in cardiac muscle. One is at CRUs that contain DHPRs and RyRs. In these cardiac CRUs, RyR2 and a1c DHPR are in proximity of each other, but not closely linked, so that they may not have a direct molecular interaction. A second location of RyR2 is on SR cisternae that are not attached to surface membrane/T tubules. The RyR2 in these cisternae, which are often several microns away from any DHPRs, must necessarily be activated indirectly. <![CDATA[<b>Novel model of calcium and inositol 1,4,5-trisphosphate regulation of InsP<sub>3</sub> receptor channel gating in native endoplasmic reticulum</b>]]> https://scielo.conicyt.cl/scielo.php?script=sci_arttext&pid=S0716-97602004000400004&lng=en&nrm=iso&tlng=en The InsP3R Ca2+-release channel has biphasic dependence on cytoplasmic free Ca2+ concentration ([Ca2+]i). InsP3 activates gating primarily by reducing high [Ca2+]i inhibition. To determine whether relieving Ca2+ inhibition is sufficient for activation, we examined single-channels in low [Ca2+]i in the absence of InsP3 by patch clamping isolated Xenopus oocyte nuclei. For both endogenous Xenopus type 1 and recombinant rat type 3 InsP3R channels, spontaneous InsP3-independent activities with low open probability Po (~ 0.03) were observed in [Ca2+]i < 5 nM, whereas none were observed in 25 nM Ca2+. These results establish the half-maximal inhibitory [Ca2+]i in the absence of InsP3 and demonstrate that the channel can be active when all of its ligand-binding sites are unoccupied. In the simplest allosteric model that fits all observations in nuclear patch-clamp studies, the tetrameric channel can adopt six conformations, the equilibria among which are controlled by two inhibitory, one activating Ca2+-binding, and one InsP3-binding sites in a manner similar to the Monod-Wyman-Changeux model. InsP3 binding activates gating by affecting the relative affinity for Ca2+ of one of the inhibitory sites in different channel conformations, transforming it into an activating site. Ca2+ inhibition of InsP3-liganded channels is mediated by an InsP3-independent second inhibitory site. <![CDATA[<b>Phosphorylation of Ryanodine Receptors</b>]]> https://scielo.conicyt.cl/scielo.php?script=sci_arttext&pid=S0716-97602004000400005&lng=en&nrm=iso&tlng=en Both cardiac and skeletal muscle ryanodine receptors (RyRs) are parts of large complexes that include a number of kinases and phosphatases. These RyRs have several potential phosphorylation sites in their cytoplasmic domains, but the functional consequences of phosphorylation and the identity of the enzymes responsible have been subjects of considerable controversy. Hyperphosphorylation of Ser-2809 in RyR2 (cardiac isoform) and Ser-2843 in RyR1 (skeletal isoform) has been suggested to cause the dissociation of the FK506-binding protein (FKBP) from RyRs, producing "leaky channels," but some laboratories find no relationship between phosphorylation and FKBP binding. Also debated is the identity of the kinases that phosphorylate these serines: cAMP-dependent protein kinase (PKA) versus calmodulin kinase II (CaMKII). Phosphorylation of other targets of these kinases could also alter calcium homeostasis. For example, PKA also phosphorylates phospholamban (PLB), altering the Sarco-endoplasmic reticulum Ca2+ ATPase (SERCA) activity. This review summarizes the major findings and controversies associated with phosphorylation of RyRs. <![CDATA[<b>The interaction of ryanoids with individual ryanodine receptor channels</b>]]> https://scielo.conicyt.cl/scielo.php?script=sci_arttext&pid=S0716-97602004000400006&lng=en&nrm=iso&tlng=en Ryanodine binds with high affinity and specificity to a class of Ca2+-release channels known as ryanodine receptors (RyR). The interaction with RyR results in a dramatic alteration in function with open probability (Po) increasing markedly and rates of ion translocation modified. We have investigated the features of ryanodine that govern the interaction of the ligand with RyR and the mechanisms underlying the subsequent alterations in function by monitoring the effects of congeners and derivatives of ryanodine (ryanoids) on individual RyR2 channels. While the interaction of all tested ryanoids results in an increased Po, the amplitude of the modified conductance state depends upon the structure of the ryanoid. We propose that different rates of cation translocation observed in the various RyR-ryanoid complexes represent different conformations of the channel stabilized by specific conformers of the ligand. On the time scale of a single channel experiment ryanodine binds irreversibly to the channel. However, alterations in structure yield some ryanoids with dissociation rate constants orders of magnitude greater than ryanodine. The probability of occurrence of the RyR-ryanoid complex is sensitive to trans-membrane voltage, with the vast majority of the influence of potential arising from a voltage-driven alteration in the affinity of the ryanoid-binding site. <![CDATA[<b>Redox regulation of RyR-mediated Ca<sup>2+</sup> release in muscle and neurons </b>]]> https://scielo.conicyt.cl/scielo.php?script=sci_arttext&pid=S0716-97602004000400007&lng=en&nrm=iso&tlng=en Changes in the redox state of the intracellular ryanodine receptor/Ca2+ release channels of skeletal and cardiac muscle or brain cortex neurons affect their activity. In particular, agents that oxidize or alkylate free SH residues of the channel protein strongly enhance Ca2+-induced Ca2+ release, whereas reducing agents have the opposite effects. We will discuss here how modifications of highly reactive cysteine residues by endogenous redox agents or cellular redox state influence RyR channel activation by Ca2+ and ATP or inhibition by Mg2+. Possible physiological and pathological implications of these results on cellular Ca2+ signaling will be addressed as well. <![CDATA[<b>Inositol 1,4,5-trisphosphate receptors in the heart </b>]]> https://scielo.conicyt.cl/scielo.php?script=sci_arttext&pid=S0716-97602004000400008&lng=en&nrm=iso&tlng=en Inositol 1,4,5-trisphosphate (InsP3) is an established calcium-mobilizing messenger, which is well-known to activate Ca2+ signaling in many cell types. Contractile cardiomyocytes express hormone receptors that are coupled to the production of InsP3. Such cardioactive hormones, including endothelin, may have profound inotropic and arrhythmogenic actions, but it is unclear whether InsP3 underlies any of these effects. We have examined the expression and localization of InsP3 receptors (InsP3Rs), and the potential role of InsP3 in modulating cardiac excitation-contraction coupling (EC coupling). Stimulation of electrically-paced atrial and ventricular myocytes with a membrane-permeant InsP3 ester was found to evoke an increase in the amplitudes of action potential-evoked Ca2+ transients and to cause pro-arrhythmic diastolic Ca2+ transients. All the effects of the InsP3 ester could be blocked using a membrane-permeant antagonist of InsP3Rs (2-aminoethoxydiphenyl borate; 2-APB). Furthermore, 2-APB blocked arrhythmias evoked by endothelin and delayed the onset of positive inotropic responses. Our data indicate that atrial and ventricular cardiomyocytes express functional InsP3Rs, and these channels have the potential to influence EC coupling. <![CDATA[<b><i>To be or not to be…</i></b><b> Nicotinic acid adenine dinucleotide phosphate (NAADP): A new intracellular second messenger?</b>]]> https://scielo.conicyt.cl/scielo.php?script=sci_arttext&pid=S0716-97602004000400009&lng=en&nrm=iso&tlng=en Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent activator of intracellular Ca2+ release in several vertebrate and invertebrate systems. The role of the NAADP system in physiologicalprocesses is being extensively investigated at the present time. The NAADP receptor and its associated Ca2+ pool have been hypothesized to be important in several physiologicalprocesses including fertilization, T cell activation, and pancreaticsecretion. However, whether NAADP is a new second messenger ora tool for the discovery of a new Ca2+ channel is still an unansweredquestion. Research developed over the last two years has provided some important clues to whether NAADP is or not a physiological cellular messenger. In this short review, I will discuss some of these new findings that are helping us to find an answer to the important question: Is NAADP a second messenger or not? <![CDATA[<B>Functional equivalence of dihydropyridine receptor a1S and b1a subunits in triggering excitation-contraction coupling in skeletal muscle</B>]]> https://scielo.conicyt.cl/scielo.php?script=sci_arttext&pid=S0716-97602004000400010&lng=en&nrm=iso&tlng=en Molecular understanding of the mechanism of excitation-contraction (EC) coupling in skeletal muscle has been made possible by cultured myotube models lacking specific dihydropyridine receptor (DHPR) subunits and ryanodine receptor type 1 (RyR1) isoforms. Transient expression of missing cDNAs in mutant myotubes leads to a rapid recovery, within days, of various Ca2+ current and EC coupling phenotypes. These myotube models have thus permitted structure-function analysis of EC coupling domains present in the DHPR controlling the opening of RyR1. The purpose of this brief review is to highlight advances made by this laboratory towards understanding the contribution of domains present in a1S and b1a subunits of the skeletal DHPR to EC coupling signaling. Our main contention is that domains of the a1S II-III loop are necessary but not sufficient to recapitulate skeletal-type EC coupling. Rather, the structural unit that controls the EC coupling signal appears to be the a1S/b1a pair <![CDATA[<b>Calmodulin and Calcium-release Channels</b>]]> https://scielo.conicyt.cl/scielo.php?script=sci_arttext&pid=S0716-97602004000400011&lng=en&nrm=iso&tlng=en Calmodulin (CaM) is a ubiquitous cytosolic protein that plays a critical role in regulating cellular functions by altering the activity of a large number of ion channels. There are many examples for CaM directly mediating the feedback effects of Ca2+ on Ca2+ channels. Recently the molecular mechanisms by which CaM interacts with voltage-gated Ca2+ channels, Ca2+-activated K+ channels and ryanodine receptors have been clarified. CaM plays an important role in regulating these ion channels through lobe-specific Ca2+ detection. CaM seems to behave as a channel subunit. It binds at low [Ca2+] and undergoes conformational changes upon binding of Ca2+, leading to an interaction with another part of the channel to regulate its gating. Here we focus on the mechanism by which CaM regulates the inositol 1,4,5-trisphosphate receptor (IP3R). Although the IP3R is inhibited by CaM and by other CaM-like proteins in the presence of Ca2+, we conclude that CaM does not act as the Ca2+ sensor for IP3R function. Furthermore we discuss a novel Ca2+-induced Ca2+-release mechanism found in A7r5 (embryonic rat aorta) and 16HBE14o- (human bronchial mucosa) cells for which CaM acts as a Ca2+ sensor <![CDATA[<b>Control of dual isoforms of Ca<sup>2+</sup> release channels in muscle</b>]]> https://scielo.conicyt.cl/scielo.php?script=sci_arttext&pid=S0716-97602004000400012&lng=en&nrm=iso&tlng=en Here we compare excitation-contraction coupling in single muscle cells of frogs and rats. Because amphibians have isoform 3 (or b) of the ryanodine receptor/Ca2+ release channel, in addition to 1 (a), which is also present in the mammal, any extra feature present in the frog may in principle be attributed to isoform 3. Ca2+ release under voltage clamp depolarization has a peak and a steady phase in both taxonomic classes, but the peak is more marked in the frog, where the ratio of amplitudes of the two phases is voltage-dependent. This dependence is a hallmark of CICR. Confocal imaging identified Ca2+ sparks in the frog, but not in the voltage-clamped rat cells. Because Ca2+ sparks involve CICR both observations indicate that the contribution of CICR is minor or null in the mammal. The "couplon" model well accounts for observations in the frog, but assumes a structure that we now know to be valid only for the rat. A revised model is proposed, whereby the isoform 3 channels, located parajunctionally, are activated by CICR and contribute its characteristic global and local features. Several issues regarding the roles of different channels remain open to further study. <![CDATA[<b>Modulation of cytosolic calcium signaling by protein kinase A-mediated phosphorylation of inositol 1,4,5-trisphosphate receptors</b>]]> https://scielo.conicyt.cl/scielo.php?script=sci_arttext&pid=S0716-97602004000400013&lng=en&nrm=iso&tlng=en Calcium release via intracellular Ca2+ release channels is a central event underpinning the generation of numerous, often divergent physiological processes. In electrically non-excitable cells, this Ca2+ release is brought about primarily through activation of inositol 1,4,5-trisphosphate receptors and typically takes the form of calcium oscillations. It is widely believed that information is carried in the temporal and spatial characteristics of these signals. Furthermore, stimulation of individual cells with different agonists can generate Ca2+ oscillations with dramatically different spatial and temporal characteristics. Thus, mechanisms must exist for the acute regulation of Ca2+ release such that agonist-specific Ca2+ signals can be generated. One such mechanism by which Ca2+ signals can be modulated is through simultaneous activation of multiple second messenger pathways. For example, activation of both the InsP3 and cAMP pathways leads to the modulation of Ca2+ release through protein kinase A mediated phosphoregulation of the InsP3R. Indeed, each InsP3R subtype is a potential substrate for PKA, although the functional consequences of this phosphorylation are not clear. This review will focus on recent advances in our understanding of phosphoregulation of InsP3R, as well as the functional consequences of this modulation in terms of eliciting specific cellular events. <![CDATA[<b>Modulation of sarcoplasmic reticulum calcium release by calsequestrin in cardiac myocytes</b>]]> https://scielo.conicyt.cl/scielo.php?script=sci_arttext&pid=S0716-97602004000400014&lng=en&nrm=iso&tlng=en Calsequestrin (CASQ2) is a high capacity Ca-binding protein expressed inside the sarcoplasmic reticulum (SR). Mutations in the cardiac calsequestrin gene (CASQ2) have been linked to arrhythmias and sudden death induced by exercise and emotional stress. We have studied the function of CASQ2 and the consequences of arrhythmogenic CASQ2 mutations on intracellular Ca signalling using a combination of approaches of reverse genetics and cellular physiology in adult cardiac myocytes. We have found that CASQ2 is an essential determinant of the ability of the SR to store and release Ca2+ in cardiac muscle. CASQ2 serves as a reservoir for Ca2+ that is readily accessible for Ca2+-induced Ca2+ release (CICR) and also as an active Ca2+ buffer that modulates the local luminal Ca-dependent closure of the SR Ca2+ release channels. At the same time, CASQ2 stabilizes the CICR process by slowing the functional recharging of SR Ca2+ stores. Abnormal restitution of the Ca2+ release channels from a luminal Ca-dependent refractory state could account for ventricular arrhythmias associated with mutations in the CASQ2 gene. <![CDATA[<b>Regulation of cardiac excitation-contraction coupling by sorcin, a novel modulator of ryanodine receptors</b>]]> https://scielo.conicyt.cl/scielo.php?script=sci_arttext&pid=S0716-97602004000400015&lng=en&nrm=iso&tlng=en Activation of Ca2+ release channels/ryanodine receptors (RyR) by the inward Ca2+ current (I Ca) gives rise to Ca2+-induced Ca2+ release (CICR), the amplifying Ca2+ signaling mechanism that triggers contraction of the heart. CICR, in theory, is a high-gain, self-regenerating process, but an unidentified mechanism stabilizes it in vivo. Sorcin, a 21.6 kDa Ca2+-binding protein, binds to cardiac RyRs with high affinity and completely inhibits channel activity. Sorcin significantly inhibits both the spontaneous activity of RyRs in quiescent cells (visualized as Ca2+ sparks) and the I Ca-triggered activity of RyRs that gives rise to [Ca2+]i transients. Since sorcin decreases the amplitude of the [Ca2+]i transient without affecting the amplitude of I Ca, the overall effect of sorcin is to reduce the "gain" of excitation-contraction coupling. Immunocytochemical staining shows that sorcin localizes to the dyadic space of ventricular cardiac myocytes. Ca2+ induces conformational changes and promotes translocation of sorcin between soluble and membranous compartments, but the [Ca2+] required for the latter process (ED50 = ~200 mM) appears to be reached only within the dyadic space. Thus, sorcin is a potent inhibitor of both spontaneous and I Ca-triggered RyR activity and may play a role in helping terminate the positive feedback loop of CICR. <![CDATA[<b>Peptide and protein modulation of local Ca<sup>2+ </sup>release events in permeabilized skeletal muscle fibers</b>]]> https://scielo.conicyt.cl/scielo.php?script=sci_arttext&pid=S0716-97602004000400016&lng=en&nrm=iso&tlng=en Local discrete elevations in myoplasmic Ca2+ (Ca2+ sparks) arise from the opening of a small group of RyRs. Summation of a large number of Ca2+ sparks gives rise to the whole cell Ca2+ transient necessary for muscle contraction. Unlike sarcoplasmic reticulum vesicle preparations and isolated single channels in artificial membranes, the study of Ca2+ sparks provides a means to understand the regulation of a small group of RyRs in the environment of a functionally intact triad and in the presence of endogenous regulatory proteins. To gain insight into the mechanisms that regulate the gating of RyRs we have utilized laser scanning confocal microscopy to measure Ca2+ sparks in permeabilized frog skeletal muscle fibers. This review summarizes our recent studies using both exogenous (ImperatoxinA and domain peptides) and endogenous (calmodulin) modulators of RyR to gain insight into the number of RyR Ca2+ release channels underlying a Ca2+ spark, how domain-domain interactions within RyR regulate the functional state of the channel as well as gating mechanisms of RyR in living muscle fibers <![CDATA[<b>Ca<sup>2+</sup> entry, efflux and release in smooth muscle</b>]]> https://scielo.conicyt.cl/scielo.php?script=sci_arttext&pid=S0716-97602004000400017&lng=en&nrm=iso&tlng=en Control of smooth muscle is vital for health. The major route to contraction is a rise in intracellular [Ca2+], determined by the entry and efflux of Ca2+ and release and re-uptake into the sarcoplasmic reticulum (SR). We review these processes in myometrium, to better understand excitation-contraction coupling and develop strategies for preventing problematic labours. The main mechanism of elevating [Ca2+] is voltage-gated L-type channels, due to pacemaker activity, which can be modulated by agonists. The rise of [Ca2+] produces Ca-calmodulin and activates MLCK. This phosphorylates myosin and force results. Without Ca2+ entry uterine contraction fails. The Na/Ca exchanger (NCX) and plasma membrane Ca-ATPase (PMCA) remove Ca2+, with contributions of 30% and 70% respectively. Studies with PMCA-4 knockout mice show that it contributes to reducing [Ca2+] and relaxation. The SR contributes to relaxation by vectorially releasing Ca2+ to the efflux pathways, and thereby increasing their rates. Agonists binding produces IP3 which can release Ca from the SR but inhibition of SR Ca2+ release increases contractions and Ca2+ transients. It is suggested that SR Ca2+ targets K+ channels on the surface membrane and thereby feedback to inhibit excitability and contraction. <![CDATA[<b>Possible link of different slow calcium signals generated by membrane potential and hormones to differential gene expression in cultured muscle cells</b>]]> https://scielo.conicyt.cl/scielo.php?script=sci_arttext&pid=S0716-97602004000400018&lng=en&nrm=iso&tlng=en 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. <![CDATA[<b>IP<sub>3</sub> receptors and Ca<sup>2+</sup> signals in adult skeletal muscle satellite cells <i>in situ</i></b>]]> https://scielo.conicyt.cl/scielo.php?script=sci_arttext&pid=S0716-97602004000400019&lng=en&nrm=iso&tlng=en In this short article we review muscle satellite cell characteristics and our studies in adult rodent muscle satellite cells in situ. Using confocal laser scanning microscopy and immunocytochemistry, a high level of IP3 receptor (IP3R) immunostaining was detected in satellite cells. These cells were identified by their peripheral position, their size, the shape of their nucleus, the paucity of the apparent cytoplasm, and the immunostaining with specific molecular markers such as a-actinin, the neural cell adhesion molecule (N-CAM) and desmin. High extracellular K+ (60 mM) induced long-lasting Ca2+ signalsin satellite cells in situ. We suggest that electrical activity stimulates IP3-associated Ca2+ signals that could act in concert with signaling pathways triggered by growth factors and/or hormones <![CDATA[<b>Regulation of capacitative and non-capacitative Ca<sup>2+</sup> entry in A7r5 vascular smooth muscle cells</b>]]> https://scielo.conicyt.cl/scielo.php?script=sci_arttext&pid=S0716-97602004000400020&lng=en&nrm=iso&tlng=en A capacitative Ca2+ entry (CCE) pathway, activated by depletion of intracellular Ca2+ stores, is thought to mediate much of the Ca2+ entry evoked by receptors that stimulate phospholipase C (PLC). However, in A7r5 vascular smooth muscle cells, vasopressin, which stimulates PLC, empties intracellular Ca2+ stores but simultaneously inhibits their ability to activate CCE. The diacylglycerol produced with the IP3 that empties the stores is metabolized to arachidonic and this leads to activation of nitric oxide (NO) synthase, production of NO and cyclic GMP, and consequent activation of protein kinase G. The latter inhibits CCE. In parallel, NO directly activates a non-capacitative Ca2+ entry (NCCE) pathway, which is entirely responsible for the Ca2+ entry that occurs in the presence of vasopressin. This reciprocal regulation of two Ca2+ entry pathways ensures that there is sequential activation of first NCCE in the presence of vasopressin, and then a transient activation of CCE when vasopressin is removed. We suggest that the two routes for Ca2+ entry may selectively direct Ca2+ to processes that mediate activation and then recovery of the cell. <![CDATA[<b>1</b><b>a</b><b>,25(OH)<sub>2</sub>D<sub>3 </sub>induces capacitative calcium entry involving a trpc3 protein in skeletal muscle and osteoblastic cells</b>]]> https://scielo.conicyt.cl/scielo.php?script=sci_arttext&pid=S0716-97602004000400021&lng=en&nrm=iso&tlng=en This work describes the involvement of TRPC proteins in capacitative calcium entry (CCE) induced by 1a,25-dihydroxy-vitamin-D3 [1a,25(OH)2D3] in chick skeletal muscle and in rat osteoblast-like cells (ROS 17/2.8) and the role of the vitamin D receptor (VDR) in this non-genomic rapid response mediated by the hormone. We propose that an endogenous TRPC3 protein mediates 1a,25(OH)2D3 modulation of CCE in these cells, which seems to implicate VDR-TRPC3 association and the participation of an INAD-like scaffold protein. <![CDATA[<b>Mitochondrial Ca<sup>2+ </sup>homeostasis in health and disease </b>]]> https://scielo.conicyt.cl/scielo.php?script=sci_arttext&pid=S0716-97602004000400022&lng=en&nrm=iso&tlng=en Although it has long been known that mitochondria possess a complex molecular repertoire for accumulating and releasing Ca2+, only in recent years has a large body of data demonstrated that these organelles promptly respond to Ca2+-mediated cell stimulations. In this contribution, we will review the principles of mitochondrial Ca2+ homeostasis and its signaling role in different physiological and pathological conditions. <![CDATA[<b>Local and global Ca<sup>2+</sup> signals: physiology and pathophysiology </b>]]> https://scielo.conicyt.cl/scielo.php?script=sci_arttext&pid=S0716-97602004000400023&lng=en&nrm=iso&tlng=en The pancreatic acinar unit is a classical example of a polarized tissue. Even in isolation, these cells retain their polarity, and this has made them particularly useful for Ca2+ signaling studies. In 1990, we discovered that this cell has the capability of producing both local cytosolic and global Ca2+ signals. The mechanisms underlying this signal generation have now been established. Furthermore, it has become clear that the local signals are sufficient for the control of both fluid and enzyme secretion, whereas prolonged global signals are dangerous and give rise to acute pancreatitis, a disease where the pancreas digests itself. <![CDATA[<B>Interplay between ER Ca<SUP>2+</SUP> uptake and release fluxes in neurons and its impact on [Ca<SUP>2+</SUP>] dynamics </B>]]> https://scielo.conicyt.cl/scielo.php?script=sci_arttext&pid=S0716-97602004000400024&lng=en&nrm=iso&tlng=en In neurons, depolarizing stimuli open voltage-gated Ca2+ channels, leading to Ca2+ entry and a rise in the cytoplasmic free Ca2+ concentration ([Ca2+]i). While such [Ca2+]i elevations are initiated by Ca2+ entry, they are also influenced by Ca2+ transporting organelles such as mitochondria and the endoplasmic reticulum (ER). This review summarizes contributions from the ER to depolarization-evoked [Ca2+]i responses in sympathetic neurons. As in other neurons, ER Ca2+ uptake depends on SERCAs, while passive Ca2+ release depends on ryanodine receptors (RyRs). RyRs are Ca2+ permeable channels that open in response to increases in [Ca2+]i, thereby permitting [Ca2+]i elevations to trigger Ca2+ release through Ca2+-induced Ca2+ release (CICR). However, whether this leads to net Ca2+ release from the ER critically depends upon the relative rates of Ca2+ uptake and release. We found that when RyRs are sensitized with caffeine, small evoked [Ca2+]i elevations do trigger net Ca2+ release, but in the absence of caffeine, net Ca2+ uptake occurs, indicating that Ca2+ uptake is stronger than Ca2+ release under these conditions. Nevertheless, by increasing ER Ca2+ permeability, RyRs reduce the strength of Ca2+ buffering by the ER in a [Ca2+]I-dependent manner, providing a novel mechanism for [Ca2+]i response acceleration. Analysis of the underlying Ca2+ fluxes provides an explanation of this and two other modes of CICR that are revealed as [Ca2+]i elevations become progressively larger <![CDATA[<b>Imaging single-channel calcium microdomains by total internal reflection microscopy </b>]]> https://scielo.conicyt.cl/scielo.php?script=sci_arttext&pid=S0716-97602004000400025&lng=en&nrm=iso&tlng=en The microdomains of Ca2+ in the cytosol around the mouth of open Ca2+ channels are the basic `building blocks' from which cellular Ca2+ signals are constructed. Moreover, the kinetics of local [Ca2+] closely reflect channel gating, so their measurement holds promise as an alternative to electrophysiological patch-clamp recording as a means to study single channel behavior. We have thus explored the development of optical techniques capable of imaging single-channel Ca2+ signals with good spatial and temporal resolution, and describe results obtained using total internal reflection fluorescence microscopy to monitor Ca2+ influx through single N-type channels expressed in Xenopus oocytes <![CDATA[<B>The Gating of Polycystin Signaling Complex </B>]]> https://scielo.conicyt.cl/scielo.php?script=sci_arttext&pid=S0716-97602004000400026&lng=en&nrm=iso&tlng=en Mutations in either polycystin-2 (PC2) or polycystin-1 (PC1) proteins cause severe, potentially lethal, kidney disorders (autosomal dominant polycystic kidney disease, ADPKD) and multiple extrarenal disease phenotypes. PC2, a member of the transient receptor potential channel superfamily and PC1, an orphan membrane receptor of largely unknown function, are thought to be part of a common signalling pathway. Here, I show that co-assembly of full-length PC1 with PC2 forms an ion channel signalling complex in which PC1 regulates PC2 channel gating through a structural rearrangement of the polycystin complex (Delmas et al., 2004a). These polycystin complexes function either as a receptor-cation channel or as a G-protein-coupled receptor. Thus, PC1 acts as a prototypical membrane receptor that regulates G-proteins and plasmalemmal PC2, a bimodal mechanism that may account for the multifunctional roles of polycystin proteins in various cell types. Genetic alteration of polycystin proteins such as those occurring in kidney diseases may impede polycystin signalling, thereby providing a likely mechanistic explanation to the pathogenesis of ADPKD. Our proposed mechanism may also be paradigmatic for the function of polycystin orthologues and other polycystin-related proteins in a variety of nonrenal cell types, including sperm, muscle cells and sensory neurons <![CDATA[<b>Endoplasmic reticulum calcium signaling in nerve cells </b>]]> https://scielo.conicyt.cl/scielo.php?script=sci_arttext&pid=S0716-97602004000400027&lng=en&nrm=iso&tlng=en The endoplasmic reticulum (ER) is an important organelle involved in various types of signaling in nerve cells. The ER serves as a dynamic Ca2+ pool being thus involved in rapid signaling events associated with cell stimulation by either electrical (action potential) or chemical (neurotransmitters) signals. This function is supported by Ca2+ release channels (InsP3 and ryanodine receptors) and SERCA Ca2+ pumps residing in the endomembrane. In addition the ER provides a specific environment for the posttranslational protein processing and transport of various molecules towards their final destination. In parallel, the ER acts as a "calcium tunnel," which facilitates Ca2+ movements within the cell by avoiding cytoplasmic routes. Finally the ER appears as a source of numerous signals aimed at the nucleus and involved in long-lasting adaptive cellular responses. All these important functions are controlled by intra-ER free Ca2+ which integrates various signaling events and establishes a link between fast signaling, associated with ER Ca2+ release/uptake, and long-lasting adaptive responses relying primarily on the regulation of protein synthesis. Disruption of ER Ca2+ homeostasis triggers several forms of cellular stress response and is intimately involved in neurodegeneration and neuronal cell death <![CDATA[<b>Signal transduction and gene expression regulated by calcium release from internal stores in excitable cells</b>]]> https://scielo.conicyt.cl/scielo.php?script=sci_arttext&pid=S0716-97602004000400028&lng=en&nrm=iso&tlng=en Calcium regulation of several transcription factors involves different calcium-dependent signaling cascades and engages cytoplasmic as well as nuclear calcium signals. The study of the specific sources of calcium signals involved in regulation of gene expression in skeletal muscle has been addressed only recently. In this tissue, most cytoplasmic and nuclear calcium signals originate from calcium release from internal stores, mediated either by ryanodine receptor (RyR) or IP3 receptor (IP3R) channels. The latter are located both in the sarcoplasmic reticulum (SR) and in the nuclear membrane, and their activation results in long-lasting nuclear calcium increase. The calcium signals mediated by RyR and IP3R are very different in kinetics, amplitude and subcellular localization; an open question is whether these differences are differentially sensed by transcription factors. In neurons, it is well established that calcium entry through L-type calcium channels and NMDA receptors plays a role in the regulation of gene expression. Increasing evidence, however, points to a role for calcium release from intracellular stores in this process. In this article, we discuss how RyR-mediated calcium release contributes to the activation of the calcium-dependent transcription factor CREB and the subsequent LTP generation. We present novel results from our laboratory showing ERK-mediated CREB activation by hydrogen peroxide. This activation takes place in the absence of extracellular calcium and is blocked by inhibitory ryanodine concentrations, suggesting it is caused by redox activation of RyR-mediated calcium release.