Aldosterone- and testosterone-mediated intracellular calcium response in skeletal muscle cell cultures

Fast nongenomic steroid actions in several cell types seem to be mediated by second messengers such as intracellular calcium ([Ca(2+)](i)) and inositol 1,4,5-trisphosphate (IP(3)). We have shown the presence of both slow calcium transients and IP(3) receptors associated with cell nuclei in cultured skeletal muscle cells. The effect of steroids on [Ca(2+)](i) was monitored in Fluo 3-acetoxymethyl ester-loaded myotubes by either confocal microscopy or fluorescence microscopy, with the use of out-of-focus fluorescence elimination. The mass of IP(3) was determined by radioreceptor displacement assay. [Ca(2+)](i) changes after either aldosterone (10-100 nM) or testosterone (50-100 nM) were observed; a relatively fast (<2 min) calcium transient, frequently accompanied by oscillations, was evident with both hormones. A slow rise in [Ca(2+)](i) that reached its maximum after a 30-min exposure to aldosterone was also observed. Calcium responses seem to be fairly specific for aldosterone and testosterone, because several other steroid hormones do not induce detectable changes in fluorescence, even at 100-fold higher concentrations. The mass of IP(3) increased transiently to reach two- to threefold the basal level 45 s after addition of either aldosterone or testosterone, and the IP(3) transient was more rapid than the fast calcium signal. Spironolactone, an inhibitor of the intracellular aldosterone receptor, or cyproterone acetate, an inhibitor of the testosterone receptor, had no effect on the fast [Ca(2+)](i) signal or in the increase in IP(3) mass. These signals could mean that there are distinct nongenomic pathways for the action of these two steroids in skeletal muscle cells.     

No evidence for inositol 1,4,5-trisphosphate-dependent Ca2+ release in isolated fibers of adult mouse skeletal muscle

The presence and role of functional inositol 1,4,5-trisphosphate (IP(3)) receptors (IP(3)Rs) in adult skeletal muscle are controversial. The current consensus is that, in adult striated muscle, the relative amount of IP(3)Rs is too low and the kinetics of Ca(2+) release from IP(3)R is too slow compared with ryanodine receptors to contribute to the Ca(2+) transient during excitation-contraction coupling. However, it has been suggested that IP(3)-dependent Ca(2+) release may be involved in signaling cascades leading to regulation of muscle gene expression. We have reinvestigated IP(3)-dependent Ca(2+) release in isolated flexor digitorum brevis (FDB) muscle fibers from adult mice. Although Ca(2+) transients were readily induced in cultured C2C12 muscle cells by (a) UTP stimulation, (b) direct injection of IP(3), or (c) photolysis of membrane-permeant caged IP(3), no statistically significant change in calcium signal was detected in adult FDB fibers. We conclude that the IP(3)-IP(3)R system does not appear to affect global calcium levels in adult mouse skeletal muscle.     

Role of intracellular calcium channels of nerve terminals in the regulation of mediator secretion

The review is presented, analysing the modern state of knowledge about the role of intracellularly stored calcium of nerve terminals in regulation of quantal mediator secretion in synapses. The data are considered, concerning the properties of two Ca(2+)-channels superfamilies, i.e. the ryanodine receptors (RyR) and IP3-receptors, which are incorporated into the membrane of endoplasmic reticulum fragments. The localization of cisternae, containing RyR and IP3-receptors in neurons and nerve terminals are described. The data, demonstrating the pattern of calcium signalization in neurons and terminals after their interaction with specific blockers or activators of RyRs or IP3-receptors are presented. The facts, demonstrating that calcium induced calcium release via RyRs or IP3-receptors takes part in controlling spontaneous secretion of different types of vesicles in synaptic terminals and supports the slow and fast types of regulated exocytosis of synaptic vesicles, in the course of single or repetitive activity of central or peripheral synapses are analysed.     

Sodium-dependent action potentials induced by brevetoxin-3 trigger both IP3 increase and intracellular Ca2+ release in rat skeletal myotubes

Brevetoxin-3 (PbTx-3), described to increase the open probability of voltage-dependent sodium channels, caused trains of action potentials and fast oscillatory changes in fluorescence intensity of fluo-3-loaded rat skeletal muscle cells in primary culture, indicating that the toxin increased intracellular Ca(2+) levels. PbTx-3 did not elicit calcium transients in dysgenic myotubes (GLT cell line), lacking the alpha1 subunit of the dihydropyridine receptor (DHPR), but after transfection of the alpha1DHPR cDNA to GLT cells, PbTx-3 induced slow calcium transients that were similar to those of normal cells. Ca(2+) signals evoked by PbTx-3 were inhibited by blocking either IP(3) receptors, with 2-aminoethoxydiphenyl borate, or phospholipase C with U73122. PbTx-3 caused a tetrodotoxin-sensitive increase in intracellular IP(3) mass levels, dependent on extra-cellular Na(+). A similar increase in IP(3) mass was induced by high K(+) depolarization but no action potential trains (nor calcium signals) were elicited by prolonged depolarization under current clamp conditions. The increase in IP(3) mass induced by either PbTx-3 or K(+) was also detected in Ca(2+)-free medium. These results establish that the effect of the toxin on both intracellular Ca(2+) and IP(3) levels occurs via a membrane potential sensor instead of directly by Na(+) flux and supports the notion of a train of action potentials being more efficient as a stimulus than sustained depolarization, suggesting that tetanus is the physiological stimulus for the IP(3)-dependent calcium signal involved in regulation of gene expression.     

Manipulating thyroid status alters endoplasmic reticulum calcium homeostasis in rat cerebellum

Thyroid-related hormones regulate the efficiency and expression of sarco-endoplasmic reticulum calcium ATPases in cardiac and skeletal muscle. However, little is known about the relationship between thyroid hormones and calcium (Ca2+) homeostasis in the brain. It is hypothesized that manipulating rat thyroid hormone levels would induce significant brain Ca2+ adaptations consistent with clinical findings. Adult male Sprague-Dawley rats were assigned to one of three treatment groups for 28 days: control, hypothyroid (6-n-propyl-2-thiouracil (PTU), an inhibitor of thyroxine (T4) synthesis), and hyperthyroid (T4). Throughout, rats were given weekly behavioral tests. Ca2+ accumulation decreased in the cerebellum in both hyper- and hypothyroid animals. This was specific to different ER pools of calcium with regional heterogeneity in the response to thyroid hormone manipulation. Behavioral tasks demonstrated sensitivity to thyroid manipulation, and corresponded to alterations in calcium homeostasis. Ca2+ accumulation heterogeneity in chronic hyper- and hypothyroid animals potentially explains clinical manifestations of altered thyroid status.     

Expression of the sodium/calcium exchanger in mammalian skeletal muscle cells in primary culture

Previous investigations have demonstrated molecular and functional expression, at early phases of development of skeletal muscle cells in primary culture, of cardiac isoforms of proteins involved in calcium transport and regulation, like the L-type calcium channel. Here the expression of the cardiac isoform of the Na(+)/Ca(2+) exchanger (NCX1) was studied in skeletal muscle cells developing in vitro, by using biochemical, immunological, and electrophysiological techniques. Northern and Western blot experiments revealed the presence of this cardiac exchanger and its increasing expression during the early phases of development. Confocal imaging of myotubes showed an NCX1 distribution that was predominantly sarcolemmal. The whole-cell patch-clamp technique allowed us to record ionic currents, the direction and the amplitude of which depended on extracellular sodium and calcium concentrations. The developmental changes of this functional expression could be correlated with the molecular NCX1 expression changes. Taken together these data demonstrate the presence of the NCX1 isoform of the Na(+)/Ca(2+) exchanger during in vitro myogenesis and reinforce the theory that significant levels of cardiac-type proteins are transiently expressed during the early phases of the skeletal muscle cell development.     

beta-Adrenergic receptors in rat skeletal muscle. Effects of thyroidectomy.

Thyroid hormones may participate in the regulation of beta-adrenergic receptors in skeletal muscle sarcolemmal membrane. Since skeletal muscles are not innervated by sympathetic nerve endings, the biochemical mechanism involved in the control of beta-adrenergic receptors by thyroid hormones appears to be mediated by thyroid-induced regulation of serum levels of catecholamines.     

The ryanodine receptor/calcium channel genes are widely and differentially expressed in murine brain and peripheral tissues

Ryanodine receptors (RyRs) are intracellular calcium release channels that participate in controlling cytosolic calcium levels. At variance with the probably ubiquitous inositol 1,4,5-trisphosphate-operated calcium channels (1,4,5-trisphosphate receptors), RyRs have been mainly regarded as the calcium release channels controlling skeletal and cardiac muscle contraction. Increasing evidence has recently suggested that RyRs may be more widely expressed, but this has never been extensively examined. Therefore, we cloned three cDNAs corresponding to murine RyR homologues to carry a comprehensive analysis of their expression in murine tissues. Here, we report that the three genes are expressed in almost all tissues analyzed, where tissue-specific patterns of expression were observed. In the uterus and vas deferens, expression of RyR3 was localized to the smooth muscle component of these organs. In the testis, expression of RyR1 and RyR3 was detected in germ cells. RyR mRNAs were also detected in in vitro-cultured cell lines. RyR1, RyR2, and RyR3 mRNA were detected in the cerebrum and in the cerebellum. In situ analysis revealed a cell type-specific pattern of expression in the different regions of the central nervous system. The differential expression of the three ryanodine receptor genes in the central nervous system was also confirmed using specific antibodies against the respective proteins. This widespread pattern of expression suggests that RyRs may participate in the regulation of intracellular calcium homeostasis in a range of cells wider than previously recognized.     

β-adrenergic receptors in rat skeletal muscle effects of thyroidectomy

Thyroid hormones may participate in the regulation of β-adrenergic receptors in skeletal muscle sarcolemmal membrane. Since skeletal muscles are not innervated by sympathetic nerve endings, the biochemical mechanism involved in the control of β-adrenergic receptors by thyroid hormones appears to be mediated by thyroid-induced regulation of serum levels of catecholamines.     

Differential regulation of SR calcium transporters by thyroid hormone in rat atria and ventricles

Thyroid hormone exerts positive inotropic effects on the heart mediated in part by its regulation of calcium transporter proteins, including sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA2), phospholamban (PLB), and Na(+)/Ca(2+) exchanger (NCX). To further understand the potential cardiac chamber-specific effects of thyroid hormone action, we compared the triiodo-L-thyronine (T(3)) responsiveness of calcium transporter proteins in atrial versus ventricular tissues. Rats were rendered hypothyroid by ingestion of propylthiouracil, and a subgroup of animals was treated with T(3) for 7 days (7 microg/day by constant infusion). Atrial and left ventricular (LV) tissue homogenates were analyzed for expression of SERCA2, PLB, and NCX proteins by Western blot analysis. SERCA2 protein significantly decreased by 50% in hypothyroid LV and was normalized by T(3) treatment. In contrast, SERCA2 protein in atria was unaltered in the hypothyroid state. PLB protein expression significantly increased by 1.6- and 5-fold in the hypothyroid LV and atria, respectively, and returned to euthyroid levels with T(3) treatment. Expression of NCX protein showed a greater response to T(3) treatment in atria tissue than in ventricular tissue. Sarcoplasmic reticulum calcium cycling is determined in part by the ratio of SERCA2 to PLB. This ratio was sixfold higher in the atria compared with LV, suggesting that PLB may play a minor role in the regulation of SERCA2 function in normal atria. We conclude that calcium transporter proteins are responsive to thyroid hormone in a chamber-specific manner, with atria showing a greater change in protein content in response to T(3). The differential effect on atria may account for the occurrence of atrial rather than ventricular arrhythmias in response to even mild degrees of thyrotoxicosis.     

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.     

Expression of the Na(+)/Ca(2+) exchanger in skeletal muscle

The expression of the Na(+)/Ca(2+) exchanger was studied in differentiating muscle fibers in rats. NCX1 and NCX3 isoform (Na(+)/Ca(2+) exchanger isoform) expression was found to be developmentally regulated. NCX1 mRNA and protein levels peaked shortly after birth. Conversely, NCX3 isoform expression was very low in muscles of newborn rats but increased dramatically during the first 2 wk of postnatal life. Immunocytochemical analysis showed that NCX1 was uniformly distributed along the sarcolemmal membrane of undifferentiated rat muscle fibers but formed clusters in T-tubular membranes and sarcolemma of adult muscle. NCX3 appeared to be more uniformly distributed along the sarcolemma and inside myoplasm. In the adult, NCX1 was predominantly expressed in oxidative (type 1 and 2A) fibers of both slow- and fast-twitch muscles, whereas NCX3 was highly expressed in fast glycolytic (2B) fibers. NCX2 was expressed in rat brain but not in skeletal muscle. Developmental changes in NCX1 and NCX3 as well as the distribution of these isoforms at the cellular level and in different fiber types suggest that they may have different physiological roles.     

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

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

Regulation of the calcium release channel from skeletal muscle by suramin and the disulfonated stilbene derivatives DIDS,DBDS, and DNDS

Activation of skeletal muscle ryanodine receptors (RyRs) by suramin and disulfonic stilbene derivatives (Diisothiocyanostilbene-2',2'-disulfonic acid (DIDS), 4,4'-dibenzamidostilbene-2,2'-disulfonic acid (DBDS),and 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS)) was investigated using planar bilayers. One reversible and two nonreversible mechanisms were identified. K(a) for reversible activation (approximately 100 micro M) depended on cytoplasmic [Ca(2+)] and the bilayer composition. Replacement of neutral lipids by negative phosphatidylserine increased K(a) fourfold, suggesting that reversible binding sites are near the bilayer surface. Suramin and the stilbene derivatives adsorbed to neutral bilayers with maximal mole fractions between 1-8% and with affinities approximately 100 micro M but did not adsorb to negative lipids. DIDS activated RyRs by two nonreversible mechanisms, distinguishable by their disparate DIDS binding rates (10(5) and 60 M(-1) s(-1)) and actions. Both mechanisms activated RyRs via several jumps in open probability, indicating several DIDS binding events. The fast and slow mechanisms are independent of each other, the reversible mechanism and ATP binding. The fast mechanism confers DIDS sensitivity approximately 1000-fold greater than previously reported, increases Ca(2+) activation and increases K(i) for Ca(2+)/Mg(2+) inhibition 10-fold. The slow mechanism activates RyRs in the absence of Ca(2+) and ATP, increases ATP activation without altering K(a), and slightly increases activity at pH < 6.5. These findings explain how different types of DIDS activation are observed under different conditions.     

A comparative study of ryanodine receptor (RyR) gene expression levels in a basal ray-finned fish, bichir (Polypterus ornatipinnis) and the derived euteleost zebrafish (Danio rerio)

Ryanodine receptors (RyRs) are large homotetrameric protein complexes that mediate the release of intracellular stores of calcium. Mammals possess three gene copies, RyR1, RyR2, and RyR3 that are expressed in a variety of tissue types. Teleost fish express RyR1a and RyR1b genes that are expressed in slow twitch skeletal muscle and fast twitch skeletal muscles respectively. Here we report the results of a survey of the genome of bichir (Polypterus ornatipinnis), considered the most basal ray-finned fish, for its RyR genes. The bichir genome encodes four RyR genes, RyR1a, RyR1b, RyR2, and RyR3 that phylogenetically cluster with their vertebrate orthologs. Quantitative real time PCR shows fibre type-specific expression of the RyR1a and RyR1b genes. The RyR3 gene, however, is down regulated in bichir in contrast to derived teleosts including zebrafish in which the RyR1 and RyR3 genes are co-expressed at equivalent levels.     

Differential regulation of the Na+-Ca2+ exchanger 3 (NCX3) by protein kinase PKC and PKA

Isoform 3 of the Na⁺-Ca²⁺ exchanger (NCX3) participates in the Ca²⁺ fluxes across the plasma membrane. Among the NCX family, NCX3 carries out a peculiar role due to its specific functions in skeletal muscle and the immune system and to its neuroprotective effect under stress exposure. In this context, proper understanding of the regulation of NCX3 is primordial to consider its potential use as a drug target. In this study, we demonstrated the regulation of NCX3 by protein kinase A (PKA) and C (PKC). Disparity in regulation has been previously reported among the splice variants of NCX3 therefore the activity of Ca²⁺ uptake and extrusion of the two murine variants was measured using fura-2-based Ca²⁺ imaging and revealed that both variants are similarly regulated. PKC stimulation diminished the Ca²⁺ uptake performed by NCX3 in the reverse mode, triggered by a rise in [Ca²⁺]_i or [Na⁺]_i, whereas an opposite response was observed upon PKA stimulation, with a significant increase of the Ca²⁺ uptake after a rise in [Ca²⁺]_i. The latter stimulation affected similarly the efflux capacity of NCX3 whereas Ca²⁺ extrusion capacity remained unaffected under activation of PKC. Next, using site-directed mutagenesis, the sensitivity of NCX3 to PKC was abolished by singly mutating its predicted phosphorylation sites T529 or S695. The sensitivity to PKC might be due to the influence of T529 phosphorylation on the Ca²⁺-binding domain 1. Additionally, we showed that stimulation of NCX3 by PKA occurred through residue S524. This effect may well participate in the fight-or-flight response in skeletal muscle and the long-term potentiation in hippocampus.