Cultured Rat Skeletal Muscle Research Articles

Voltage and Ca2+ activation of single large-conductance Ca2+-activated K+ channels described by a two-tiered allosteric gating mechanism.

The voltage- and Ca2+-dependent gating mechanism of large-conductance Ca2+-activated K+ (BK) channels from cultured rat skeletal muscle was studied using single-channel analysis. Channel open probability (Po) increased with depolarization, as determined by limiting slope measurements (11 mV per e-fold change in Po; effective gating charge, q(eff), of 2.3 +/- 0.6 e(o)). Estimates of q(eff) were little changed for intracellular Ca2+ (Ca2+(i)) ranging from 0.0003 to 1,024 microM. Increasing Ca2+(i) from 0.03 to 1,024 microM shifted the voltage for half maximal activation (V(1/2)) 175 mV in the hyperpolarizing direction. V(1/2) was independent of Ca2+(i) for Ca2+(i) < or = 0.03 microM, indicating that the channel can be activated in the absence of Ca2+(i). Open and closed dwell-time distributions for data obtained at different Ca2+(i) and voltage, but at the same Po, were different, indicating that the major action of voltage is not through concentrating Ca2+ at the binding sites. The voltage dependence of Po arose from a decrease in the mean closing rate with depolarization (q(eff) = -0.5 e(o)) and an increase in the mean opening rate (q(eff) = 1.8 e(o)), consistent with voltage-dependent steps in both the activation and deactivation pathways. A 50-state two-tiered model with separate voltage- and Ca2+-dependent steps was consistent with the major features of the voltage and Ca2+ dependence of the single-channel kinetics over wide ranges of Ca2+(i) (approximately 0 through 1,024 microM), voltage (+80 to -80 mV), and Po (10(-4) to 0.96). In the model, the voltage dependence of the gating arises mainly from voltage-dependent transitions between closed (C-C) and open (O-O) states, with less voltage dependence for transitions between open and closed states (C-O), and with no voltage dependence for Ca2+-binding and unbinding. The two-tiered model can serve as a working hypothesis for the Ca2+- and voltage-dependent gating of the BK channel.

Protein kinase Cdelta mediates insulin-induced glucose transport in primary cultures of rat skeletal muscle.

Insulin activates certain protein kinase C (PKC) isoforms that are involved in insulin-induced glucose transport. In this study, we investigated the possibility that activation of PKCdelta by insulin participates in the mediation of insulin effects on glucose transport in skeletal muscle. Studies were performed on primary cultures of rat skeletal myotubes. The role of PKCdelta in insulin-induced glucose uptake was evaluated both by selective pharmacological blockade and by over-expression of wild-type and point-mutated inactive PKCdelta isoforms in skeletal myotubes. We found that insulin induces tyrosine phosphorylation and translocation of PKCdelta to the plasma membrane and increases the activity of this isoform. Insulin-induced effects on translocation and phosphorylation of PKCdelta were blocked by a low concentration of rottlerin, whereas the effects of insulin on other PKC isoforms were not. This selective blockade of PKCdelta by rottlerin also inhibited insulin-induced translocation of glucose transporter 4 (GLUT4), but not glucose transporter 3 (GLUT3), and significantly reduced the stimulation of glucose uptake by insulin. When overexpressed in skeletal muscle, PKCdelta and PKCdelta were both active. Overexpression of PKCdelta induced the translocation of GLUT4 to the plasma membrane and increased basal glucose uptake to levels attained by insulin. Moreover, insulin did not increase glucose uptake further in cells overexpressing PKCdelta. Overexpression of PKCdelta did not affect basal glucose uptake or GLUT4 location. Stimulation of glucose uptake by insulin in cells overexpressing PKCdelta was similar to that in untransfected cells. Transfection of skeletal myotubes with dominant negative mutant PKCdelta did not alter basal glucose uptake but blocked insulin-induced GLUT4 translocation and glucose transport. These results demonstrate that insulin activates PKCdelta and that activated PKCdelta is a major signaling molecule in insulin-induced glucose transport.

Protein Kinase C Mediates Insulin-Induced Glucose Transport in Primary Cultures of Rat Skeletal Muscle

Protein Kinase C Mediates Insulin-Induced Glucose Transport in Primary Cultures of Rat Skeletal Muscle

Tyrosine phosphorylation of specific protein kinase C isoenzymes participates in insulin stimulation of glucose transport in primary cultures of rat skeletal muscle.

Several reports indicate that protein kinase C (PKC) plays a role in insulin-induced glucose transport in certain cells. The precise effects of insulin on specific PKC isoforms are as yet unknown. Utilizing primary cultures of rat skeletal muscle, we investigated the possibility that insulin may influence the activation state of PKC isoenzymes by inducing their translocation and tyrosine phosphorylation. This, in turn, may mediate insulin effects on glucose transport. We identified and determined the glucose transporters and PKC isoforms affected by insulin and 12-O-tetradecanoylphorbol-13-acetate (TPA). Insulin and TPA each caused an increase in glucose uptake. Insulin translocated GLUT3 and GLUT4 without affecting GLUT1. In contrast, TPA translocated GLUT1 and GLUT3 without affecting GLUT4. Insulin translocated and tyrosine phosphorylated and activated PKC-beta2 and -zeta; these effects were blocked by phosphatidylinositol 3-kinase (PI3K) inhibitors. TPA translocated and activated PKC-alpha, -beta2, and -delta; these effects were not noticeably affected by PI3K inhibitors. Furthermore, wortmannin significantly inhibited both insulin and TPA effects on GLUT translocation and glucose uptake. Finally, insulin-induced glucose transport was blocked by the specific PKC-beta2 inhibitor LY379196. These results indicate that specific PKC isoenzymes, when tyrosine-phosphorylated, are implicated in insulin-induced glucose transport in primary cultures of skeletal muscle.

Rat skeletal muscle in culture expresses the ?1 but not the ?2 protein subunit isoform of the Na+/K+ pump

Studies from this laboratory have shown that the physiological expression of the Na+/K+ pump in primary cultures of rat skeletal muscle increases with development. The molecular mechanisms underlying these changes are not known. Therefore, we have examined the expression of alpha and beta subunits of the Na+/K+ pump at both the protein and mRNA levels during myogenesis of primary skeletal muscle cell cultures obtained from newborn rats. Protein isoforms were identified by Western blotting techniques with specific monoclonal and polyclonal antibodies and subunit mRNA was studied with specific cDNA probes. Freshly isolated skeletal muscle from newborn rats expressed both alpha1 and alpha2 protein subunits. From day 1 after plating, primary cultures expressed only the alpha1 protein isoform. In contrast, both beta1 and beta2 isoforms were expressed in freshly isolated muscle and in primary cultures, with beta1 expression being stronger in both preparations. Studies on RNA expression showed that mRNA for alpha1, alpha2, beta1, and beta2 isoforms was identified both in freshly isolated muscle and after plating of cells in culture. These findings indicate that the lack of alpha2 protein expression in primary muscle cell cultures reflects a form of posttranscriptional regulation. There did not appear to be a quantitative difference in isoform expression as a function of age or of fusion in spite of developmental increases in Na+/K+ pump activity and its dependence on cell fusion. The lack of expression of the alpha2 subunit isoform suggests that the developmental changes in physiological expression of the Na+/K+ pump in primary cultures of skeletal muscle may be attributable either to the changes in activity of the alpha1 subunit or to differential activities of alphabeta complexes involving either of the beta subunits.

Gating kinetics of single large-conductance Ca2+-activated K+ channels in high Ca2+ suggest a two-tiered allosteric gating mechanism.

The Ca2+-dependent gating mechanism of large-conductance calcium-activated K+ (BK) channels from cultured rat skeletal muscle was examined from low (4 microM) to high (1,024 microM) intracellular concentrations of calcium (Ca2+i) using single-channel recording. Open probability (Po) increased with increasing Ca2+i (K0. 5 11.2 +/- 0.3 microM at +30 mV, Hill coefficient of 3.5 +/- 0.3), reaching a maximum of approximately 0.97 for Ca2+i approximately 100 microM. Increasing Ca2+i further to 1,024 microM had little additional effect on either Po or the single-channel kinetics. The channels gated among at least three to four open and four to five closed states at high levels of Ca2+i (>100 microM), compared with three to four open and five to seven closed states at lower Ca2+i. The ability of kinetic schemes to account for the single-channel kinetics was examined with simultaneous maximum likelihood fitting of two-dimensional (2-D) dwell-time distributions obtained from low to high Ca2+i. Kinetic schemes drawn from the 10-state Monod-Wyman-Changeux model could not describe the dwell-time distributions from low to high Ca2+i. Kinetic schemes drawn from Eigen's general model for a ligand-activated tetrameric protein could approximate the dwell-time distributions but not the dependency (correlations) between adjacent intervals at high Ca2+i. However, models drawn from a general 50 state two-tiered scheme, in which there were 25 closed states on the upper tier and 25 open states on the lower tier, could approximate both the dwell-time distributions and the dependency from low to high Ca2+i. In the two-tiered model, the BK channel can open directly from each closed state, and a minimum of five open and five closed states are available for gating at any given Ca2+i. A model that assumed that the apparent Ca2+-binding steps can reach a maximum rate at high Ca2+i could also approximate the gating from low to high Ca2+i. The considered models can serve as working hypotheses for the gating of BK channels.

Discoordinate regulation of different K channels in cultured rat skeletal muscle by nerve growth factor

We investigated the effects of nerve growth factor (NGF) on expression of K+ channels in cultured skeletal muscle. The channels studied were (1) charybdotoxin (ChTx)-sensitive channels by using a polyclonal antibody raised in rabbits against ChTx, (2) Kv1.5 voltage-sensitive channels, and (3) apamin-sensitive (afterhyperpolarization) channels. Crude homogenates were prepared from cultures made from limb muscles of 1–2-day-old rat pups for identification of ChTx-sensitive and Kv1.5 channels by Western blotting techniques. Apamin-sensitive K+ channels were studied by measurement of specific [125I]-apamin binding by whole cell preparations. ChTx-sensitive channels display a fusion-related increase in expression, and NGF downregulates these channels in both myoblasts and myotubes. Voltage-dependent Kv1.5 channel expression is low in myoblasts and increases dramatically with fusion; NGF induces early expression of these channels and causes expression after fusion to increase even further. NGF downregulates apamin-sensitive channels. NGF increases the rate of fall of the action potential recorded intracellularly from single myotubes with intracellular microelectrodes. The results confirm and extend those of previous studies in showing a functional role for NGF in the regulation of membrane properties of skeletal muscle. Moreover, the findings demonstrate that the different K+ channels in this preparation are regulated in a discoordinate manner. The divergent effects of NGF on expression of different K+ channels, however, do not appear sufficient to explain the NGF-induced increase in the rate of fall of the action potential. The changes during the falling phase may rather be due to increases in channel properties or may result from an increased driving force on the membrane potential secondary to the NGF-induced hyperpolarization. J. Neurosci. Res. 56:275–283, 1999. © 1999 Wiley-Liss, Inc.

The effects of Securidaca longepedunculata root extract on ionic currents and contraction of cultured rat skeletal muscle cells

The effects of the primary extract roots of Securidaca longepedunculata were tested on sodium, calcium and potassium currents in rat skeletal muscle cells developed in culture. In addition, they were tested on depolarisation-induced contraction and resting intracellular calcium levels. S. longepedunculata extract (10 −6 g/l) increases sodium current at all potentials. No clear effect was observed on calcium current except for a slight increase at negative potentials (−30, −10 mV) revealing a 5 mV shift towards negative potentials of the I Ca/ V curve, as with potassium current. In contrast, at the same concentration, S. longepedunculata enhanced the contractile response elicited by durable depolarisation. This was not attributable to the slight increase in resting intracellular free calcium concentration which did not change during and following S. longepedunculata application. These results strongly suggest that S. longepedunculata root extract contains one or more components acting on the voltage-sensor of excitation–contraction coupling (dihydropyridine receptors), regardless of its implication as a calcium channel.

Discoordinate regulation of different K channels in cultured rat skeletal muscle by nerve growth factor.

We investigated the effects of nerve growth factor (NGF) on expression of K+ channels in cultured skeletal muscle. The channels studied were (1) charybdotoxin (ChTx)-sensitive channels by using a polyclonal antibody raised in rabbits against ChTx, (2) Kv1.5 voltage-sensitive channels, and (3) apamin-sensitive (afterhyperpolarization) channels. Crude homogenates were prepared from cultures made from limb muscles of 1-2-day-old rat pups for identification of ChTx-sensitive and Kv1.5 channels by Western blotting techniques. Apamin-sensitive K+ channels were studied by measurement of specific [125I]-apamin binding by whole cell preparations. ChTx-sensitive channels display a fusion-related increase in expression, and NGF downregulates these channels in both myoblasts and myotubes. Voltage-dependent Kv1.5 channel expression is low in myoblasts and increases dramatically with fusion; NGF induces early expression of these channels and causes expression after fusion to increase even further. NGF downregulates apamin-sensitive channels. NGF increases the rate of fall of the action potential recorded intracellularly from single myotubes with intracellular microelectrodes. The results confirm and extend those of previous studies in showing a functional role for NGF in the regulation of membrane properties of skeletal muscle. Moreover, the findings demonstrate that the different K+ channels in this preparation are regulated in a discoordinate manner. The divergent effects of NGF on expression of different K+ channels, however, do not appear sufficient to explain the NGF-induced increase in the rate of fall of the action potential. The changes during the falling phase may rather be due to increases in channel properties or may result from an increased driving force on the membrane potential secondary to the NGF-induced hyperpolarization.

The beta subunit increases the Ca2+ sensitivity of large conductance Ca2+-activated potassium channels by retaining the gating in the bursting states.

Coexpression of the beta subunit (KV,Cabeta) with the alpha subunit of mammalian large conductance Ca2+- activated K+ (BK) channels greatly increases the apparent Ca2+ sensitivity of the channel. Using single-channel analysis to investigate the mechanism for this increase, we found that the beta subunit increased open probability (Po) by increasing burst duration 20-100-fold, while having little effect on the durations of the gaps (closed intervals) between bursts or on the numbers of detected open and closed states entered during gating. The effect of the beta subunit was not equivalent to raising intracellular Ca2+ in the absence of the beta subunit, suggesting that the beta subunit does not act by increasing all the Ca2+ binding rates proportionally. The beta subunit also inhibited transitions to subconductance levels. It is the retention of the BK channel in the bursting states by the beta subunit that increases the apparent Ca2+ sensitivity of the channel. In the presence of the beta subunit, each burst of openings is greatly amplified in duration through increases in both the numbers of openings per burst and in the mean open times. Native BK channels from cultured rat skeletal muscle were found to have bursting kinetics similar to channels expressed from alpha subunits alone.

Kinetic structure of large-conductance Ca2+-activated K+ channels suggests that the gating includes transitions through intermediate or secondary states. A mechanism for flickers.

Mechanisms for the Ca2+-dependent gating of single large-conductance Ca2+-activated K+ channels from cultured rat skeletal muscle were developed using two-dimensional analysis of single-channel currents recorded with the patch clamp technique. To extract and display the essential kinetic information, the kinetic structure, from the single channel currents, adjacent open and closed intervals were binned as pairs and plotted as two-dimensional dwell-time distributions, and the excesses and deficits of the interval pairs over that expected for independent pairing were plotted as dependency plots. The basic features of the kinetic structure were generally the same among single large-conductance Ca2+-activated K+ channels, but channel-specific differences were readily apparent, suggesting heterogeneities in the gating. Simple gating schemes drawn from the Monod- Wyman-Changeux (MWC) model for allosteric proteins could approximate the basic features of the Ca2+ dependence of the kinetic structure. However, consistent differences between the observed and predicted dependency plots suggested that additional brief lifetime closed states not included in MWC-type models were involved in the gating. Adding these additional brief closed states to the MWC-type models, either beyond the activation pathway (secondary closed states) or within the activation pathway (intermediate closed states), improved the description of the Ca2+ dependence of the kinetic structure. Secondary closed states are consistent with the closing of secondary gates or channel block. Intermediate closed states are consistent with mechanisms in which the channel activates by passing through a series of intermediate conformations between the more stable open and closed states. It is the added secondary or intermediate closed states that give rise to the majority of the brief closings (flickers) in the gating.

Time-irreversible subconductance gating associated with Ba2+ block of large conductance Ca2+-activated K+ channels.

Ba2+ block of large conductance Ca2+-activated K+ channels was studied in patches of membrane excised from cultures of rat skeletal muscle using the patch clamp technique. Under conditions in which a blocking Ba2+ ion would dissociate to the external solution (150 mM N-methyl-D-glucamine+o, 500 mM K+i, 10 microM Ba2+i, +30 mV, and 100 microM Ca2+i to fully activate the channel), Ba2+ blocks with a mean duration of approximately 2 s occurred, on average, once every approximately 100 ms of channel open time. Of these Ba2+ blocks, 78% terminated with a single step in the current to the fully open level and 22% terminated with a transition to a subconductance level at approximately 0.26 of the fully open level (preopening) before stepping to the fully open level. Only one apparent preclosing was observed in approximately 10,000 Ba2+ blocks. Thus, the preopenings represent Ba2+-induced time-irreversible subconductance gating. The fraction of Ba2+ blocks terminating with a preopening and the duration of preopenings (exponentially distributed, mean = 0.75 ms) appeared independent of changes in [Ba2+]i or membrane potential. The fractional conductance of the preopenings increased from 0.24 at +10 mV to 0.39 at +90 mV. In contrast, the average subconductance level during normal gating in the absence of Ba2+ was independent of membrane potential, suggesting different mechanisms for preopenings and normal subconductance levels. Preopenings were also observed with 10 mM Ba2+o and no added Ba2+i. Adding K+, Rb+, or Na+ to the external solution decreased the fraction of Ba2+ blocks with preopenings, with K+ and Rb+ being more effective than Na+. These results are consistent with models in which the blocking Ba2+ ion either induces a preopening gate, and then dissociates to the external solution, or moves to a site located on the external side of the Ba2+ blocking site and acts directly as the preopening gate.

Role of Serine/Threonine Protein Phosphatases in Insulin Regulation of Na+/K+-ATPase Activity in Cultured Rat Skeletal Muscle Cells

In this study, we examined the potential role of serine/threonine protein phosphatase-1 (PP-1) and PP-2A in the mechanism of Na+/K+-ATPase activation by insulin in the rat skeletal muscle cell line L6. Incubation of L6 cells with insulin caused a time- and dose-dependent stimulation of ouabain-sensitive plasma membrane Na+/K+-ATPase activity. Pretreatment with okadaic acid (OA; 0.1-1 microM) or calyculin A (1 microM) blocked insulin's effect on Na+/K+-ATPase activation. Low concentrations of OA that specifically inhibit PP-2A were ineffective. Immunoprecipitation of the enzyme from 32P-labeled cells with an antibody directed against the alpha-1 subunit of the enzyme revealed a 60% decrease in 110-kDa protein phosphorylation in insulin-treated cells. The presence of calyculin A blocked insulin-mediated dephosphorylation of Na+/K+-ATPase, whereas low concentrations of OA were ineffective. To further confirm the role of PP-1, we used L6 cell lines that overexpress the glycogen/SR-associated regulatory subunit of PP-1, PP-1G. Overexpression of PP-1G resulted in a 3-fold increase in insulin-stimulated PP-1 catalytic activity. This was accompanied by a 30% increase in basal Na+/K+-ATPase activity and a >2-fold increase in insulin's effect on pump activity. Inhibition of phosphatidylinositol-3 kinase with wortmannin blocked insulin-stimulated PP-1 activation as well as the dephosphorylation and activation of Na+/K+-ATPase. We conclude that insulin regulates the activity of Na+/K+-ATPase by promoting dephosphorylation of the alpha subunit via an insulin-stimulated PP-1 and that phosphatidylinositol-3 kinase-generated signals may mediate insulin activation of PP-1 and Na+/K+-ATPase.

Inhibition of Cholesterol Synthesis by Squalene Synthase Inhibitors Does Not Induce Myotoxicityin Vitro

The cholesterol-lowering HMG CoA reductase inhibitors (HMGRI), pravastatin and lovastatin, have been associated with skeletal myopathy in humans and in rats. In a previousin vitrostudy, HMGRI-induced changes in neonatal rat skeletal muscle cells were characterized by reversible inhibition of protein synthesis and loss of differentiated myotubes at concentrations markedly lower than those inducing enzyme leakage. Myotoxicity was determined to be directly related to inhibition of HMG CoA reductase, since mevalonate, the immediate product of HMG CoA reductase metabolism, abrogated the drug-induced changes. Farnesol, geranylgeraniol, and squalene are metabolites of mevalonate. Squalene, formed from farnesol by squalene synthase, is the first metabolite solely committed to cholesterol synthesis. In contrast, geranylgeraniol, formed by the addition of an isoprene group to farnesol, is the first metabolite uncommitted to cholesterol synthesis. The objective of the present study was to determine the role of inhibition of cholesterol synthesis in HMGRI-inducedin vitromyotoxicity. HMGRI-treated neonatal rat skeletal muscle cultures were supplemented with farnesol and geranylgeraniol, and in another study, muscle cultures were exposed to two squalene synthase inhibitors (SSI), BMS-187745 and its prodrug ester, BMS-188494. Endpoints evaluated for both studies included protein synthesis ([3H]leucine incorporation), total cellular protein (a measure of cell loss), intra- and extracellular lactate dehydrogenase activity (a measure of membrane integrity), cholesterol biosynthesis ([14C]acetate incorporation), and morphology. HMG CoA reductase inhibitor-induced morphologic changes and inhibition of protein synthesis were significantly ameliorated by supplementation with farnesol and geranylgeraniol. In contrast to HMGRI-inducedin vitromyotoxicity, SSI induced an irreversible, minimal cytotoxicity at close to maximum soluble concentrations. These results indicate that depletion of metabolites of geranylgeranyl pyrophosphate, and not inhibition of cholesterol synthesis, is the primary cause of HMG CoA reductase-induced myotoxicity.

The effect of modulating the glycogen-associated regulatory subunit of protein phosphatase-1 on insulin action in rat skeletal muscle cells.

Recent studies from this laboratory have shown that insulin rapidly stimulates a membranous protein phosphatase-1 (PP-1) in cultured rat skeletal muscle cells and isolated rat adipocytes. Stimulation of PP-1 is accompanied by the phosphorylation of a 160-kDa regulatory subunit of PP-1 (PP-1G). To further evaluate the exact role of this subunit in insulin action, L6 rat skeletal muscle cells were stably transfected with a vector containing the gene for PP-1G in the sense and antisense orientations. Transfection with the vector containing the PP-1G gene in the sense orientation yielded three stable clones with a 4- to 6-fold increase in PP-1G protein expression compared to those of wild-type L6 cells and neo control cells harboring an empty expression vector. Compared to the neo control, overexpression of PP-1G resulted in a 3-fold increase in insulin-stimulated PP-1 catalytic activity bound to PP-1G immunoprecipitates. These cell lines were examined for insulin's effect on glucose uptake, glycogen synthase activity, and glycogen synthesis. Insulin treatment resulted in an approximately 2-fold increase in 2-deoxyglucose uptake in recombinant cells compared to control cells (P < 0.05). This increase in 2-deoxyglucose transport was accompanied by an approximately 2-fold increase in insulin-stimulated glycogen synthase fractional activity (P < 0.05) and a 2- to 4-fold increase in insulin-stimulated glycogen synthesis compared to control cells. In conjunction with these observations, we found that an 85% depletion of endogenous PP-1G, using antisense constructs, resulted in a complete lack of PP-1 activation and an inhibition of basal and insulin-stimulated glucose transport. We conclude that the PP-1G holoenzyme is the major phosphatase regulated by insulin in vivo and plays an important role in insulin-stimulated glycogen synthesis by regulating the catalytic activity of bound PP-1.

Interferon-γ Alters Nerve-Induced Redistribution of Acetylcholine Receptors in Cultured Rat Skeletal Muscle Cells

The influence of recombinant interferon-γ (rIFN-γ) on the development of acetylcholine receptor (AChR) aggregates in cocultures of rat embryonic muscle cells and spinal cord neurons was studied by counting the number of AChR aggregates in relation to cholinergic nerve fibers coming to the muscle fibers. rIFN-γ caused no decrease in the number of cholinergic nerve fibers, but inhibited the increase in the number of AChR aggregates that occurs early during cocultivation and is an early sign in the development of neuromuscular junctions. rIFN-γ stimulated release of nitric oxide, but no effects on aggregation of AChRs occurred after exposure to a nitric oxide synthase inhibitor, l-NG-monomethylarginine, or by the addition of nitroprusside, a generator of nitric oxide. No effect was seen on the number of AChR aggregates when the cultures were exposed to rIFN-γ at later time points of cocultivation, when the increase in number of AChRs had already occurred. These studies indicate that the key immunoregulatory cytokine IFN-γ can cause alterations in the early process of synapse formation and that these effects are independent of the nitric oxide release caused by the cytokine.

Cytotoxic and non-cytotoxic effects of the MEIC reference chemicals on spontaneously contracting primary cultured rat skeletal muscle cells

This study was designed to evaluate the suitability of a multi-endpoint test system using primary cultured spontaneously contracting rat skeletal muscle cells to indicate an acute neuro- and/or cardiotoxic potential of chemicals. The concentration-dependent effects of the 50 MEIC (Multicenter Evaluation of In Vitro Cytotoxicity) reference chemicals on contractility, indicative of the functional integrity of the electrically active muscle cell membrane, were determined. Additionally, effects on two other endpoints, glucose consumption and viability, were monitored to reveal whether alterations in contractility were associated with cytotoxicity. In total, 30 of the tested compounds inhibited contractility at non-cytotoxic concentrations. The contractility-inhibiting and cytotoxic potencies differed by factors of more than 10 in the case of diazepam, phenol, propranolol, phenobarbital, mercuric chloride, thioridazine, verapamil, chloroquine, quinidine, phenytoin and atropine, of more than 100 in the case of amitriptyline, dextropropoxyphene, orphenadrine and amphetamine, and even more than 1000 for nicotine. On the basis of the available knowledge of the acute toxic effects and modes of acute toxic action of the test compounds, this characteristic response pattern is shown to be highly predictive for compounds reported to be cardio- and/or neurotoxic owing to interference with excitable membrane functions and/or neurotransmission.

High Ca2+ concentrations induce a low activity mode and reveal Ca2(+)-independent long shut intervals in BK channels from rat muscle.

1. Large-conductance calcium-activated K+ channels (BK channels) often display long closed intervals at higher levels of Ca2+. To gain further insight into possible mechanisms for these intervals, currents were recorded from single BK channels, using the patch clamp technique, from patches of membrane excised from primary cultures of rat skeletal muscle. 2. High intracellular calcium concentrations ([Ca2+]i; 10-1000 microM) induced a low activity mode and revealed isolated long shut intervals. Neither of these phenomena were due to the Ba2+ that typically contaminates reagent grade salts. 3. The low activity mode was characterized by typically single brief open intervals with mean durations of 0.1 ms, separated by long shut intervals with mean durations of 100 ms. The very low open probability of about 0.001 during the low activity mode would make a sojourn to this mode functionally equivalent to a sojourn to an inactive state. The durations of sojourns in the low activity mode were exponentially distributed, with the mean durations ranging from about 1 s in 10 microM Ca(i)2+, to 4.5 s in 1000 microM Ca(i)2+. With increased filtering, the brief open intervals would escape detection so that a sojourn to the low activity mode would appear as a single shut interval. A typical channel spent less than 5% of its time in the low activity mode for [Ca2+]i < 10 microM. This increased to about 30% for [Ca2+]i > 100-1000 microM. A kinetic model with three closed states and two open states could approximate the gating of the low activity mode. 4. The isolated long shut intervals were not from the low activity mode, suggesting a different underlying mechanism. Their frequency of occurrence of about 0.3 s-1 did not increase with increasing [Ca2+]i, indicating that they did not arise from a slow Ca2+ block. Their durations were exponentially distributed, with a mean of 127 ms, which was independent of [Ca2+]i, suggesting that a single Ca(2+)-independent closed state or block underlies the isolated long shut intervals. At higher [Ca2+]i, up to 60% of the shut time could be spent in the isolated long shut intervals. 5. These observations suggest that activation of BK channels by high [Ca2+]i can be limited by sojourns to a low activity mode and also by isolated long shut intervals, two additional phenomena that will have to be accounted for in the gating of BK channels.

Effect of tumor necrosis factor-alpha on insulin action in cultured rat skeletal muscle cells.

In this study, the acute effects of tumor necrosis factor (TNF)-alpha on insulin-stimulated glucose uptake, glycogen synthesis, and protein phosphatase-1 (PP-1) activation were examined in cultured rat skeletal muscle cell line, L6. Exposure of L6 cells to low concentrations of TNF-alpha (10 ng/ml for 60 min) inhibited basal and insulin stimulated 2-deoxyglucose uptake (40-50% decrease in basal and insulin stimulated glucose uptake respectively, when compared with controls, P < 0.05). The effect of TNF-alpha was more pronounced when the incubation period was extended to 6 and 12 h. TNF-alpha also blocked insulin activation of glycogen synthase (GS) and inhibited glycogen synthesis (measured as [14C]-glucose incorporated into glycogen). Because GS is activated by dephosphorylation via protein phosphatase-1 (PP-1), we examined the effect of TNF- alpha on PP-1 activation. As reported by us earlier (Srinivasan, M., and N. Begum, J Biol Chem 269:16662-16667, 1994), insulin rapidly stimulated PP-1 and concomitantly inhibited PP-2A activities in L6 cells. Pretreatment with TNF- alpha for 10-60 min blocked subsequent insulin-induced activation of PP-1. The impaired activation of PP-1 was accompanied by a reduction in insulin-stimulated phosphorylation of the regulatory subunit of PP-1. cAMP-Rp diastereomer, a cAMP antagonist failed to prevent the detrimental effects of TNF-alpha on PP-1. Cell permeable ceramide analogs, C2, C6, and Sphingomyelinase mimicked the effects of TNF-alpha on PP-1 inhibition. Furthermore, TNF-alpha treatment was accompanied by an increase in cellular ceramide levels, with concomitant reductions in sphingomyelin. We conclude that TNF-alpha blocks insulin-stimulated glycogen synthesis by inhibiting PP-1 activation via ceramide release.

Effect of tumor necrosis factor-alpha on insulin action in cultured rat skeletal muscle cells

Effect of tumor necrosis factor-alpha on insulin action in cultured rat skeletal muscle cells