The endoplasmic reticulum (ER) is a dynamic intracellular organelle with multiple functions essential for cellular homeostasis, development, and stress responsiveness. In response to cellular stress, a well-established signaling cascade, the unfolded protein response (UPR), is activated. This intricate mechanism is an important means of reestablishing cellular homeostasis and alleviating the inciting stress. Now, emerging evidence has demonstrated that the UPR influences cellular metabolism through diverse mechanisms, including calcium and lipid transfer, raising the prospect of involvement of these processes in the pathogenesis of disease, including neurodegeneration, cancer, diabetes mellitus and cardiovascular disease. Here, we review the distinct functions of the ER and UPR from a metabolic point of view, highlighting their association with prevalent pathologies.

Involvement of intracellular Ca(2+) and ERK1/2 phosphorylation in the fast nongenomic effects of androgens in myotubes was investigated. Testosterone or nandrolone produced fast (<1 min) and transient increases in intracellular Ca(2+) with an oscillatory pattern. Calcium signals were slightly reduced in Ca(2+)-free medium, but lack of oscillations was evident. Signals were blocked by U-73122 and xestospongin B, inhibitors of inositol 1,4,5-trisphosphate (IP(3)) pathway. Furthermore, IP(3) increased transiently 2- to 3-fold 45 sec after hormone addition. Cyproterone neither affected the fast Ca(2+) signal nor the increase in IP(3). Calcium increases could also be induced by the impermeant testosterone conjugated to BSA, and the effect of testosterone was abolished in cells incubated with guanosine 5'-O-(2-thiodiphosphate) or pertussis toxin. Stimulation of myotubes with testosterone, nandrolone, or testosterone conjugated to BSA increased immunodetectable phosphorylation of ERK1/2 within 5 min, and this effect was not inhibited by cyproterone. Phosphorylation was blocked by the use of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethylester, U-73122, and xestospongin B as well as by dominant negative Ras, MAPK kinase (MEK), or the MEK inhibitor PD-98059. In addition, guanosine 5'-O-(2-thiodiphosphate) or pertussis toxin blocked ERK1/2 phosphorylation. These results are consistent with a fast effect of testosterone, involving a G protein-linked receptor at the plasma membrane, IP(3)-mediated Ca(2+) signal, and the Ras/MEK/ERK pathway in muscle cells.

Ceramides stimulate mitochondrial fission and this event is associated with early activation of cardiomyocyte apoptosis.

ATP released from cells is known to activate plasma membrane P2X (ionotropic) or P2Y (metabotropic) receptors. In skeletal muscle cells, depolarizing stimuli induce both a fast calcium signal associated with contraction and a slow signal that regulates gene expression. Here we show that nucleotides released to the extracellular medium by electrical stimulation are partly involved in the fast component and are largely responsible for the slow signals. In rat skeletal myotubes, a tetanic stimulus (45 Hz, 400 1-ms pulses) rapidly increased extracellular levels of ATP, ADP, and AMP after 15 s to 3 min. Exogenous ATP induced an increase in intracellular free Ca 2؉ concentration, with an EC 50 value of 7.8 ؎ 3.1 M. Exogenous ADP, UTP, and UDP also promoted calcium transients. Both fast and slow calcium signals evoked by tetanic stimulation were inhibited by either 100 M suramin or 2 units/ml apyrase. Apyrase also reduced fast and slow calcium signals evoked by tetanus (45 Hz, 400 0.3-ms pulses) in isolated mouse adult skeletal fibers. A likely candidate for the ATP release pathway is the pannexin-1 hemichannel; its blockers inhibited both calcium transients and ATP release. The dihydropyridine receptor co-precipitated with both the P2Y 2 receptor and pannexin-1. As reported previously for electrical stimulation, 500 M ATP significantly increased mRNA expression for both c-fos and interleukin 6. Our results suggest that nucleotides released during skeletal muscle activity through pannexin-1 hemichannels act through P2X and P2Y receptors to modulate both Ca 2؉ homeostasis and muscle physiology.

Inositol 1,4,5-trisphosphate (IP(3)) receptors (IP(3)R) and ryanodine receptors (RyR) were localized in cultured rodent muscle fractions by binding of radiolabeled ligands (IP(3) and ryanodine), and IP(3)R were visualized in situ by fluorescence immunocytological techniques. Also explored was the effect of K(+) depolarization on IP(3) mass and Ca(2+) transients studied using a radio-receptor displacement assay and fluorescence imaging of intracellular fluo 3. RyR were located in a microsomal fraction; IP(3)R were preferentially found in the nuclear fraction. Fluorescence associated with anti-IP(3)R antibody was found in the region of the nuclear envelope and in a striated pattern in the sarcoplasmic areas. An increase in external K(+) affected membrane potential and produced an IP(3) transient. Rat myotubes displayed a fast-propagating Ca(2+) signal, corresponding to the excitation-contraction coupling transient and a much slower Ca(2+) wave. Both signals were triggered by high external K(+) and were independent of external Ca(2+). Slow waves were associated with cell nuclei and were propagated leaving "glowing" nuclei behind. Different roles are proposed for at least two types of Ca(2+) release channels, each mediating an intracellular signal in cultured skeletal muscle.

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Controlled generation of reactive oxygen species (ROS) may contribute to physiological intracellular signaling events. We determined ROS generation in primary cultures of rat skeletal muscle after field stimulation (400 1-ms pulses at a frequency of 45 Hz) or after depolarization with 65 mM K þ for 1 min. Both protocols induced a long lasting increase in dichlorofluorescein fluorescence used as ROS indicator. Addition of diphenyleneiodonium (DPI), an inhibitor of NAD(P)H oxidase, PEG-catalase, a ROS scavenger, or nifedipine, an inhibitor of the skeletal muscle voltage sensor, significantly reduced this increase. Myotubes contained both the p47 phox and gp91 phox phagocytic NAD(P)H oxidase subunits, as revealed by immunodetection. To study the effects of ROS, myotubes were exposed to hydrogen peroxide (H 2 O 2 ) at concentrations (100-200 mM) that did not alter cell viability; H 2 O 2 induced a transient intracellular Ca 2þ rise, measured as fluo-3 fluorescence. Minutes after Ca 2þ signal initiation, an increase in ERK1/2 and CREB phosphorylation and of mRNA for the early genes c-fos and c-jun was detected. Inhibition of ryanodine receptor (RyR) decreased all effects induced by H 2 O 2 and NAD(P)H oxidase inhibitors DPI and apocynin decreased ryanodine-sensitive calcium signals. Activity-dependent ROS generation is likely to be involved in regulation of calcium-controlled intracellular signaling pathways in muscle cells.

The dihydropyridine receptor (DHPR), normally a voltage-dependent calcium channel, functions in skeletal muscle essentially as a voltage sensor, triggering intracellular calcium release for excitation-contraction coupling. In addition to this fast calcium release, via ryanodine receptor (RYR) channels, depolarization of skeletal myotubes evokes slow calcium waves, unrelated to contraction, that involve the cell nucleus (Jaimovich, E., R. Reyes, J.L. Liberona, and J.A. Powell. 2000. Am. J. Physiol. Cell Physiol. 278:C998–C1010). We tested the hypothesis that DHPR may also be the voltage sensor for these slow calcium signals. In cultures of primary rat myotubes, 10 μM nifedipine (a DHPR inhibitor) completely blocked the slow calcium (fluo-3-fluorescence) transient after 47 mM K+ depolarization and only partially reduced the fast Ca2+ signal. Dysgenic myotubes from the GLT cell line, which do not express the α1 subunit of the DHPR, did not show either type of calcium transient following depolarization. After transfection of the α1 DNA into the GLT cells, K+ depolarization induced slow calcium transients that were similar to those present in normal C2C12 and normal NLT cell lines. Slow calcium transients in transfected cells were blocked by nifedipine as well as by the G protein inhibitor, pertussis toxin, but not by ryanodine, the RYR inhibitor. Since slow Ca2+ transients appear to be mediated by IP3, we measured the increase of IP3 mass after K+ depolarization. The IP3 transient seen in control cells was inhibited by nifedipine and was absent in nontransfected dysgenic cells, but α1-transfected cells recovered the depolarization-induced IP3 transient. In normal myotubes, 10 μM nifedipine, but not ryanodine, inhibited c-jun and c-fos mRNA increase after K+ depolarization. These results suggest a role for DHPR-mediated calcium signals in regulation of early gene expression. A model of excitation-transcription coupling is presented in which both G proteins and IP3 appear as important downstream mediators after sensing of depolarization by DHPR.