Fibroblast growth factor-21 potentiates glucose transport in skeletal muscle fibers.

Fibroblast growth factor 21 (FGF21) is a pleiotropic peptide hormone that is considered a myokine playing a role in a variety of endocrine functions, including regulation of glucose transport and lipid metabolism. Although FGF21 has been associated with glucose metabolism in skeletal muscle cells, its cellular mechanism in adult skeletal muscle fibers glucose uptake is poorly understood. In the present study, we found that FGF21 induced a dose-response effect, increasing glucose uptake in skeletal muscle fibers from flexor digitorum brevis muscle of mice, evaluated using the fluorescent glucose analog 2-NBDG (300 µM) in single living fibers. This effect was prevented by the use of either Cytochalasin B (5 µM) or Indinavir (100 µM), both antagonists of GLUT4 activity. The use of PI3K inhibitors such as Wortmannin (100 nM) and LY294002 (50 µM) completely prevented the FGF21-dependent glucose uptake. In fibers electroporated with the construct encoding GLUT4myc-eGFP chimera and stimulated with FGF21 (100 ng/mL), a strong sarcolemmal GLUT4 label was detected. This effect promoted by FGF21 was demonstrated to be dependent on atypical PKC-ζ, by using selective PKC inhibitors. FGF21 at low concentrations potentiated the effect of insulin on glucose uptake but at high concentrations, completely inhibited the uptake in the presence of insulin. These results suggest that FGF21 regulates glucose uptake by a mechanism mediated by GLUT4 and dependent on atypical PKC-ζ- in skeletal muscle.

Membrane Cholesterol in Skeletal Muscle: A Novel Player in Excitation-Contraction Coupling and Insulin Resistance.

Membrane cholesterol is critical for signaling processes in a variety of tissues. We will address here current evidence supporting an emerging role of cholesterol on excitation-contraction coupling and glucose transport in skeletal muscle. We have centered our review on the transverse tubule system, a complex network of narrow plasma membrane invaginations that propagate membrane depolarization into the fiber interior and allow nutrient delivery into the fibers. We will discuss current evidence showing that transverse tubule membranes have remarkably high cholesterol levels and we will address how modifications of cholesterol content influence excitation-contraction coupling. In addition, we will discuss how membrane cholesterol levels affect glucose transport by modulating the insertion into the membrane of the main insulin-sensitive glucose transporter GLUT4. Finally, we will address how the increased membrane cholesterol levels displayed by obese animals, which also present insulin resistance, affect these two particular skeletal muscle functions.

Calcium signaling in insulin action on striated muscle.

Striated muscles (skeletal and cardiac) are major physiological targets of insulin and this hormone triggers complex signaling pathways regulating cell growth and energy metabolism. Insulin increases glucose uptake into muscle cells by stimulating glucose transporter (GLUT4) translocation from intracellular compartments to the cell surface. The canonical insulin-triggered signaling cascade controlling this process is constituted by well-mapped tyrosine, lipid and serine/threonine phosphorylation reactions. In parallel to these signals, recent findings reveal insulin-dependent Ca(2+) mobilization in skeletal muscle cells and cardiomyocytes. Specifically, insulin activates the sarco-endoplasmic reticulum (SER) channels that release Ca(2+) into the cytosol i.e., the Ryanodine Receptor (RyR) and the inositol 1,4,5-triphosphate receptor (IP3R). In skeletal muscle cells, a rapid, insulin-triggered Ca(2+) release occurs through RyR, that is brought about upon S-glutathionylation of cysteine residues in the channel by reactive oxygen species (ROS) produced by the early activation of the NADPH oxidase (NOX2). In cardiomyocytes insulin induces a fast and transient increase in cytoplasmic [Ca(2+)]i trough L-type Ca(2+) channels activation. In both cell types, a relatively slower Ca(2+) release also occurs through IP3R activation, and is required for GLUT4 translocation and glucose uptake. The insulin-dependent Ca(2+) released from IP3R of skeletal muscle also promotes mitochondrial Ca(2+) uptake. We review here these actions of insulin on intracellular Ca(2+) channel activation and their impact on GLUT4 traffic in muscle cells, as well as other implications of insulin-dependent Ca(2+) release from the SER.

Exercise sensitizes skeletal muscle to extracellular ATP for IL-6 expression in mice.

Active skeletal muscle synthesizes and releases interleukin-6 (IL-6), which plays important roles in the organism's adaptation to exercise. Autocrine/paracrine ATP signaling has been shown to modulate IL-6 expression. The aim of this study was to determine whether a period of physical activity modifies the ATP-induced IL-6 expression. BalbC mice were either subject to 5 weeks voluntary wheel running (VA) or kept sedentary (SED). Flexor digitorum brevis muscles were dissected, stimulated with different ATP concentrations (0-100 µM) and IL-6 mRNA levels were measured using qPCR. ATP evoked a concentration-dependent rise in IL-6 mRNA in both SED and VA mice. VA mice however, had significantly higher ATP sensitivity (pD2 pharmacological values: VA=5.58±0.02 vs. SED=4.95±0.04, p<0.05). Interestingly, in VA mice we observed a positive correlation between the level of physical activity and the IL-6 mRNA increase following fiber stimulation with 10 µM ATP. In addition, there were lower P2Y2- and higher P2Y14-receptor mRNA levels in skeletal muscles of VA compared to SED mice, showing plasticity of nucleotide receptors with exercise. These results suggest that exercise increases skeletal muscle ATP sensitivity, a response dependent on the level of physical activity performed. This could have an important role in the mechanisms controlling skeletal muscle adaptation to exercise and training.

Anabolic androgenic steroids and intracellular calcium signaling: a mini review on mechanisms and physiological implications.

Increasing evidence suggests that nongenomic effects of testosterone and anabolic androgenic steroids (AAS) operate concertedly with genomic effects. Classically, these responses have been viewed as separate and independent processes, primarily because nongenomic responses are faster and appear to be mediated by membrane androgen receptors, whereas long-term genomic effects are mediated through cytosolic androgen receptors regulating transcriptional activity. Numerous studies have demonstrated increases in intracellular Ca2+ in response to AAS. These Ca2+ mediated responses have been seen in a diversity of cell types, including osteoblasts, platelets, skeletal muscle cells, cardiac myocytes and neurons. The versatility of Ca2+ as a second messenger provides these responses with a vast number of pathophysiological implications. In cardiac cells, testosterone elicits voltage-dependent Ca2+ oscillations and IP3R-mediated Ca2+ release from internal stores, leading to activation of MAPK and mTOR signaling that promotes cardiac hypertrophy. In neurons, depending upon concentration, testosterone can provoke either physiological Ca2+ oscillations, essential for synaptic plasticity, or sustained, pathological Ca2+ transients that lead to neuronal apoptosis. We propose therefore, that Ca2+ acts as an important point of crosstalk between nongenomic and genomic AAS signaling, representing a central regulator that bridges these previously thought to be divergent responses.

An inositol 1,4,5-triphosphate (IP3)-IP3 receptor pathway is required for insulin-stimulated glucose transporter 4 translocation and glucose uptake in cardiomyocytes.

Intracellular calcium levels ([Ca2+]i) and glucose uptake are central to cardiomyocyte physiology, yet connections between them have not been studied. We investigated whether insulin regulates [Ca2+]i in cultured cardiomyocytes, the participating mechanisms, and their influence on glucose uptake via SLC2 family of facilitative glucose transporter 4 (GLUT4). Primary neonatal rat cardiomyocytes were preloaded with the Ca2+ fluorescent dye fluo3-acetoxymethyl ester compound (AM) and visualized by confocal microscopy. Ca2+ transport pathways were selectively targeted by chemical and molecular inhibition. Glucose uptake was assessed using [3H]2-deoxyglucose, and surface GLUT4 levels were quantified in nonpermeabilized cardiomyocytes transfected with GLUT4-myc-enhanced green fluorescent protein. Insulin elicited a fast, two-component, transient increase in [Ca2+]i. Nifedipine and ryanodine prevented only the first component. The second one was reduced by inositol-1,4,5-trisphosphate (IP3)-receptor-selective inhibitors (xestospongin C, 2 amino-ethoxydiphenylborate), by type 2 IP3 receptor knockdown via small interfering RNA or by transfected Gßγ peptidic inhibitor ßARKct. Insulin-stimulated glucose uptake was prevented by bis(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid-AM, 2-amino-ethoxydiphenylborate, and ßARK-ct but not by nifedipine or ryanodine. Similarly, insulin-dependent exofacial exposure of GLUT4-myc-enhanced green fluorescent protein was inhibited by bis(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid-AM and xestospongin C but not by nifedipine. Phosphatidylinositol 3-kinase and Akt were also required for the second phase of Ca2+ release and GLUT4 translocation. Transfected dominant-negative phosphatidylinositol 3-kinase γ inhibited the latter. In conclusion, in primary neonatal cardiomyocytes, insulin induces an important component of Ca2+ release via IP3 receptor. This component signals to glucose uptake via GLUT4, revealing a so-far unrealized contribution of IP3-sensitive Ca2+ stores to insulin action. This pathway may influence cardiac metabolism in conditions yet to be explored in adult myocardium.

Parallel activation of Ca(2+)-induced survival and death pathways in cardiomyocytes by sorbitol-induced hyperosmotic stress.

Hyperosmotic stress promotes rapid and pronounced apoptosis in cultured cardiomyocytes. Here, we investigated if Ca(2+) signals contribute to this response. Exposure of cardiomyocytes to sorbitol [600 mosmol (kg water)(-1)] elicited large and oscillatory intracellular Ca(2+) concentration increases. These Ca(2+) signals were inhibited by nifedipine, Cd(2+), U73122, xestospongin C and ryanodine, suggesting contributions from both Ca(2+) influx through voltage dependent L-type Ca(2+) channels plus Ca(2+) release from intracellular stores mediated by IP(3) receptors and ryanodine receptors. Hyperosmotic stress also increased mitochondrial Ca(2+) levels, promoted mitochondrial depolarization, reduced intracellular ATP content, and activated the transcriptional factor cyclic AMP responsive element binding protein (CREB), determined by increased CREB phosphorylation and electrophoretic mobility shift assays. Incubation with 1 mM EGTA to decrease extracellular [Ca(2+)] prevented cardiomyocyte apoptosis induced by hyperosmotic stress, while overexpression of an adenoviral dominant negative form of CREB abolished the cardioprotection provided by 1 mM EGTA. These results suggest that hyperosmotic stress induced by sorbitol, by increasing Ca(2+) influx and raising intracellular Ca(2+) concentration, activates Ca(2+) release from stores and causes cell death through mitochondrial function collapse. In addition, the present results suggest that the Ca(2+) increase induced by hyperosmotic stress promotes cell survival by recruiting CREB-mediated signaling. Thus, the fate of cardiomyocytes under hyperosmotic stress will depend on the balance between Ca(2+)-induced survival and death pathways.

Capacitative calcium entry in testosterone-induced intracellular calcium oscillations in myotubes.

Ca2+ oscillations are one of the most important signals within the cell. The mechanism for generation of Ca2+ oscillations is still not yet fully elucidated. We studied the role of capacitative Ca2+ entry (CCE) on intracellular Ca2+ oscillations induced by testosterone at the single-cell level in primary myotubes. Testosterone (100 nM) rapidly induced an intracellular Ca2+ rise, accompanied by Ca2+ oscillations in a majority of myotubes. Spectral analysis of the Ca2+ oscillations revealed a periodicity of 20.3 +/- 1.8 s (frequency of 49.3 +/- 4.4 mHz). In Ca(2+)-free medium, an increase in intracellular Ca2+ was still observed, but no oscillations. Neither nifedipine nor ryanodine affected the testosterone-induced Ca2+ response. This intracellular Ca2+ release was previously shown in myotubes to be dependent on inositol-1,4,5-trisphosphate (IP3). Intracellular Ca2+ store depletion in Ca(2+)-free medium, using a sarcoplasmic/endoplasmic reticulum calcium ATPase-pump inhibitor, followed by re-addition of extracellular Ca2+, gave a fast rise in intracellular Ca2+, indicating that CCE was present in these myotubes. Application of either testosterone or albumin-bound testosterone induced Ca2+ release and led to CCE after re-addition of Ca2+ to Ca(2+)-free extracellular medium. The CCE blockers 2-aminoethyl diphenylborate and La3+, as well as perturbation of the cytoskeleton by cytochalasin D, inhibited testosterone-induced Ca2+ oscillations and CCE. The steady increase in Ca2+ induced by testosterone was not, however, affected by either La3+ or cytochalasin D. These results demonstrate testosterone-induced Ca2+ oscillations in myotubes, mediated by the interplay of IP3-sensitive Ca2+ stores and Ca2+ influx through CCE.

IGF-I and insulin induce different intracellular calcium signals in skeletal muscle cells.

We studied the effect of IGF-I and insulin on intracellular Ca(2+) in primary cultured myotubes. IGF-I induced a fast and transient Ca(2+) increase, measured as fluo-3 fluorescence. This response was blocked by both genistein and AG538. IGF-I induced a fast inositol-1,4,5-trisphosphate (IP(3)) increase, kinetically similar to the Ca(2+) rise. The Ca(2+) signal was blocked by inhibitors of the IP(3) pathway. On the other hand, insulin produced a fast (<1 s) and transient Ca(2+) increase. Insulin-induced Ca(2+) increase was blocked in Ca(2+)-free medium and by either nifedipine or ryanodine. In the normal muscle NLT cell line, the Ca(2+ )signals induced by both hormones resemble those of primary myotubes. GLT cells, lacking the alpha1-subunit of dihydropyridine receptor (DHPR), responded to IGF-I but not to insulin, while GLT cells transfected with the alpha1-subunit of DHPR reacted to both hormones. Moreover, dyspedic muscle cells, lacking ryanodine receptors, responded to IGF-I as NLT cells, however they show no insulin-induced calcium increase. Moreover, G-protein inhibitors, pertussis toxin (PTX) and GDPbetaS, blocked the insulin-induced Ca(2+) increase without major modification of the response to IGF-I. The different intracellular Ca(2+) patterns produced by IGF-I and insulin may improve our understanding of the early action mechanisms for these hormones in skeletal muscle cells.

IP(3) receptor function and localization in myotubes: an unexplored Ca(2+) signaling pathway in skeletal muscle.

We present evidence for an unexplored inositol 1,4,5-trisphosphate-mediated Ca(2+) signaling pathway in skeletal muscle. RT-PCR methods confirm expression of all three known isotypes of the inositol trisphosphate receptor in cultured rodent muscle. Confocal microscopy of cultured mouse muscle, doubly labeled for inositol receptor type 1 and proteins of known distribution, reveals that the receptors are localized to the I band of the sarcoplasmic reticulum, and this staining is continuous with staining of the nuclear envelope region. These results suggest that the receptors are positioned to mediate a slowly propagating Ca(2+) wave that follows the fast Ca(2+) transient upon K(+) depolarization. This slow wave, imaged using fluo-3, resulted in an increase in nucleoplasmic Ca(2+) lasting tens of seconds, but not contraction; the slow wave was blocked by both the inositol trisphosphate receptor inhibitor 2-aminoethoxydiphenyl borate and the phospholipase C inhibitor U-73122. To test the hypothesis that these slow Ca(2+) signals are involved in signal cascades leading to regulation of gene expression, we assayed for early effects of K(+) depolarization on mitogen-activated protein kinases, specifically extracellular-signal related kinases 1 and 2 and the transcription factor cAMP response element-binding protein (CREB). Within 30-60 seconds following depolarization, phosphorylation of both the kinases and CREB was evident and could be inhibited by 2-aminoethoxydiphenyl borate. These results suggest a signaling system mediated by Ca(2+) and inositol trisphosphate that could regulate gene expression in muscle cells.

Low threshold T-type calcium current in rat embryonic chromaffin cells.

1. The gating kinetics and functions of low threshold T-type current in cultured chromaffin cells from rats of 19-20 days gestation (E19-E20) were studied using the patch clamp technique. Exocytosis induced by calcium currents was monitored by the measurement of membrane capacitance and amperometry with a carbon fibre sensor. 2. In cells cultured for 1-4 days, the embryonic chromaffin cells were immunohistochemically identified by using polyclonal antibodies against dopamine beta-hydroxylase (DBH) and syntaxin. The immuno-positive cells could be separated into three types, based on the recorded calcium current properties. Type I cells showed exclusively large low threshold T-type current, Type II cells showed only high voltage activated (HVA) calcium channel current and Type III cells showed both T-type and HVA currents. These cells represented 44 %, 46 % and 10 % of the total, respectively. 3. T-type current recorded in Type I cells became detectable at -50 mV, reached its maximum amplitude of 6.8 +/- 1.2 pA pF(-1) (n = 5) at -10 mV and reversed around +50 mV. The current was characterized by criss-crossing kinetics within the -50 to -30 mV voltage range and a slow deactivation (deactivation time constant, tau(d) = 2 ms at -80 mV). The channel closing and inactivation process included both voltage-dependent and voltage-independent steps. The antihypertensive drug mibefradil (200 nM) reduced the current amplitude to about 65 % of control values. Ni(2+) also blocked the current in a dose-dependent manner with an IC(50) of 25 microM. 4. T-type current in Type I cells did not induce exocytosis, while catecholamine secretion by exocytosis could be induced by HVA calcium current in both Type II and Type III cells. The failure to induce exocytosis by T-type current in Type I cells was not due to insufficient Ca(2+) influx through the T-type calcium channel. 5. We suggest that T-type current is expressed in developing immature chromaffin cells. The T-type current is replaced progressively by HVA calcium current during pre- and post-natal development accompanying the functional maturation of the exocytosis mechanism.

Calcium transients in 1B5 myotubes lacking ryanodine receptors are related to inositol trisphosphate receptors.

Potassium depolarization of skeletal myotubes evokes slow calcium waves that are unrelated to contraction and involve the cell nucleus (Jaimovich, E., Reyes, R., Liberona, J. L., and Powell, J. A. (2000) Am. J. Physiol. 278, C998-C1010). Studies were done in both the 1B5 (Ry53-/-) murine "dyspedic" myoblast cell line, which does not express any ryanodine receptor isoforms (Moore, R. A., Nguyen, H., Galceran, J., Pessah, I. N., and Allen, P. D. (1998) J. Cell Biol. 140, 843-851), and C(2)C(12) cells, a myoblast cell line that expresses all three isoforms. Although 1B5 cells lack ryanodine binding, they bind tritiated inositol (1,4,5)-trisphosphate. Both type 1 and type 3 inositol trisphosphate receptors were immuno-located in the nuclei of both cell types and were visualized by Western blot analysis. After stimulation with 47 mm K(+), inositol trisphosphate mass raised transiently in both cell types. Both fast calcium increase and slow propagated calcium signals were seen in C(2)C(12) myotubes. However, 1B5 myotubes (as well as ryanodine-treated C(2)C(12) myotubes) displayed only a long-lasting, non-propagating calcium increase, particularly evident in the nuclei. Calcium signals in 1B5 myotubes were almost completely blocked by inhibitors of the inositol trisphosphate pathway: U73122, 2-aminoethoxydiphenyl borate, or xestospongin C. Results support the hypothesis that inositol trisphosphate mediates slow calcium signals in muscle cell ryanodine receptors, having a role in their time course and propagation.

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.

IP(3) receptors, IP(3) transients, and nucleus-associated Ca(2+) signals in cultured skeletal muscle.

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.

Differences in both inositol 1,4,5-trisphosphate mass and inositol 1,4,5-trisphosphate receptors between normal and dystrophic skeletal muscle cell lines.

Human normal (RCMH) and Duchenne muscular dystrophy (RCDMD) cell lines, as well as newly developed normal and dystrophic murine cell lines, were used for the study of both changes in inositol 1,4,5-trisphosphate (IP3) mass and IP3 binding to receptors. Basal levels of IP3 were increased two- to threefold in dystrophic human and murine cell lines compared to normal cell lines. Potassium depolarization induced a time-dependent IP3 rise in normal human cells and cells of the myogenic mouse cell line (129CB3), which returned to their basal levels after 60 s. However, in the human dystrophic cell line (RCDMD), IP3 levels remained high up to 200 s after potassium depolarization. Expression of IP3 receptors was studied measuring specific binding of 3H-IP3 in the murine cell lines (normal 129CB3 and dystrophic mdx XLT 4-2). All the cell lines bind 3H-IP3 with relatively high affinity (Kd: between 40 and 100 nmol/L). IP3 receptors are concentrated in the nuclear fraction, and their density is significantly higher in dystrophic cells compared to normal. These findings together with high basal levels of IP3 mass suggest a possible role for this system in the deficiency of intracellular calcium regulation in Duchenne muscular dystrophy.

Expression of ion channels during differentiation of a human skeletal muscle cell line.

An immortal, cloned cell line (RCMH), obtained from human skeletal muscle was established in our laboratory and shown to express muscle specific proteins. We measured ligand binding to ion channels, ion currents using whole cell patch clamp and intracellular calcium both in cells grown in complete media and in cells grown for 4-40 days in media supplemented with hormones and nutrients (differentiating media). Markers for differentiated muscle, such as the muscle isoform of creatine kinase and the cytoskeletal proteins alpha-actinin, alpha-sarcomeric actin, myosin and titin were present in early stages. Receptors for gamma toxin from Tityus serrulatus scorpion venom, a specific modulator for voltage dependent sodium channels, were present (0.9-1.0 pmol mg-1 protein) during stage 1 (0-6 days in culture with differentiating media) and increased by 50% in stage 3 (more than 10 days in differentiating media). High and low affinity dihydropyridine receptors present in stage 1 convert into a single type of high affinity receptors in stage 3. Both intracellular calcium release and InsP3 receptors were evident in stage 1 but ryanodine receptors were expressed only in stage 3. RCMH cells showed no voltage sensitive currents in stage 1. Between 7 and 10 days in differentiating media (stage 2), an outward potassium current was observed. Small inward currents appeared only in stage 3; we identified both tetrodotoxin sensitive and tetrodotoxin resistant sodium currents as well as calcium currents. This pattern is consistent with the expression of voltage dependent calcium release before appearance of both the action potential and ryanodine receptors.

Changes in IP3 metabolism during skeletal muscle development in vivo and in vitro.

We have investigated whether IP3 metabolism presents particular changes during critical stages of muscle development. With this aim, we have measured IP3 formation through phospholipase C activity, IP3 removal through IP3 5-phosphatase and IP3 3-kinase activities, as well as IP3 mass, during myogenesis in vivo and in vitro. In developing rat skeletal muscle, both IP3 3-kinase and 5-phosphatase activities were relatively constant from embryonary day 15, the earliest age studied to postnatal day 10; 5-phosphatase decreased upon further development. A transient, major increase in phospholipase C activity was evident at embryonary day 18 while a non-significant increase in IP3 mass was detected at this embrionary age. In rat skeletal muscle in primary culture, all enzyme activities as well as the mass of IP3 increased significantly in myotubes compared to myoblasts. Myotubes incubated with calcitonin gene-related peptide, responded with a transient increase in IP3 mass after 2 to 10 sec; the CGRP-induced increase being completely blocked by U-73122, a phospholipase C inhibitor. Furthermore, IP3 mass increased within 1 hr after exposure to differentiating agents of both RCMH cells, a line derived from normal human skeletal muscle, and C2C12 cells. These results indicate that changes in IP3 metabolism can be correlated to critical stages of muscle development and differentiation, suggesting a possible role for IP3 in these processes.

Functional muscarinic receptors in cultured skeletal muscle.

We studied the influence of muscarinic and nicotinic stimulation on both phosphoinositide metabolism and intracellular calcium levels in rat skeletal muscle primary cultures. Both nicotine and muscarine induced an increase in cytosolic calcium measured by fluo 3 fluorescence in confocal microscopy. The mass of inositol (1,4,5)trisphosphate measured by radioreceptor assay rose 2- to 3.5-fold upon carbachol, nicotine, or muscarine stimulation. The muscarine effect was mimicked by oxotremorine-M; pirenzepine prevented the muscarine-induced inositol (1,4,5)trisphosphate increase, whereas 4-diphenylacetoxy-N-methyl piperidine methiodide was ineffective. A relatively small (40 fmol/mg protein) high-affinity 3-quinuclidinylbenzilate binding to rat myotube microsomes was consistent with the muscarinic effect found. On the other hand, the effect of nicotine on the mass of inositol (1,4,5)trisphosphate was totally suppressed in sodium-free medium. Expression of Ml muscarinic receptors coupled to phospholipase C and to internal calcium stores in cultured skeletal muscle is proposed; nicotinic receptors could be acting via ion fluxes and membrane depolarization.

Voltage control of calcium transients elicited by caffeine and tetracaine in cultured rat muscle cells.

Cultured hind limb skeletal muscle cells from newborn rats were used to study the effect of caffeine and tetracaine upon intracellular Ca2+ release under voltage or current clamp conditions. Free [Ca2+]i was measured using the fluorescent calcium-sensitive dye Fluo-3. A field containing one or several myotubes was observed with a video camera and image analysis of fluorescence changes was performed. Addition of 100-500 microM tetracaine to the external saline elicited strong fluorescence responses in non-clamped cells, but significantly lower responses in cells clamped at -90 mV. At the same time, tetracaine inhibited voltage induced calcium release. Voltage and tetracaine modulation over the action of caffeine (500 microM) was also observed. Pretreatment of cells with 10 microM nifedipine abolished the caffeine induced fluorescence response in non-clamped cells. These findings suggest that, in cultured muscle cells, calcium release through the caffeine and tetracaine sensitive pathways is controlled by both membrane potential and the dihydropyridine receptor.