Nerve stimulation decreases malonyl-CoA in skeletal muscle.

This study was designed to determine the effect of in situ electrical stimulation of the sciatic nerve on malonyl-CoA, an inhibitor of carnitine palmitoyl transferase, in the gastrocnemius/plantaris muscle group of rats. The left sciatic nerve was stimulated at a frequency of 5 Hz with 100-ms trains of impulses (50 Hz) for 1, 3, or 5 min. At the end of stimulation, the left and right (nonstimulated) gastrocnemius/plantaris muscle groups were clamp-frozen and later analyzed for malonyl-CoA and other metabolites. No change was observed in the noncontracting contralateral muscles in malonyl-CoA, ATP, creatine phosphate (CP), or citrate. In the stimulated muscles, malonyl-CoA decreased from 1.7 +/- 0.1 to 1.0 +/- 0.1 nmol/g (P less than 0.05), and CP decreased from 15.8 +/- 0.9 to 12.2 +/- 1.0 mumol/g (P less than 0.05) after 3 min of stimulation. After 5 min of stimulation, malonyl-CoA was 1.0 +/- 0.1 nmol/g and CP was 10.3 +/- 1.3 mumol/g. When muscles were stimulated for 5 min with single impulses (5 Hz), malonyl-CoA was decreased from 1.8 +/- 0.3 to 1.0 +/- 0.1 nmol/g, with no change in CP, ATP, or adenosine 3',5'-cyclic monophosphate. Thus a decline in malonyl-CoA can be induced by muscle contraction independently of humoral influence.     

Mechanical stimulation improves tissue-engineered human skeletal muscle

Human bioartificial muscles (HBAMs) aretissue engineered by suspending muscle cells in collagen/MATRIGEL,casting in a silicone mold containing end attachment sites, andallowing the cells to differentiate for 8 to 16 days. The resultingHBAMs are representative of skeletal muscle in that they containparallel arrays of postmitotic myofibers; however, they differ in manyother morphological characteristics. To engineer improved HBAMs, i.e.,more in vivo-like, we developed Mechanical Cell Stimulator (MCS)hardware to apply in vivo-like forces directly to the engineeredtissue. A sensitive force transducer attached to the HBAM measuredreal-time, internally generated, as well as externally applied, forces.The muscle cells generated increasing internal forces during formationwhich were inhibitable with a cytoskeleton depolymerizer. Repetitivestretch/relaxation for 8 days increased the HBAM elasticity two- tothreefold, mean myofiber diameter 12%, and myofiber area percent 40%.This system allows engineering of improved skeletal muscle analogs aswell as a nondestructive method to determine passive force andviscoelastic properties of the resulting tissue.

     

Mechanical stimulation of skeletal muscle increases prostaglandin F2α production,cyclooxygenase activity,and cell growth by a pertussis toxin sensitive mechanism

Repetitive mechanical stimulation of differentiated skeletal muscle in tissue culture increased the long-term production of prostaglandin F_2α, an anabolic stimulator of myofiber growth. Within 4 h of initiating mechanical stimulation, the enzymatic activity of cyclooxygenase (prostaglandin GH synthase [PGHS]), a regulatory enzyme in prostaglandin synthesis, was increased 82% (P <.005), and this increase was maintained for at least 24 h. Kinetic analysis of stretch-activated cyclooxygenase activity indicated a two to threefold decrease in the enzyme's K_m, with little change in its V_max. Immunocytochemical analysis of the cell cultures indicated the presence of high levels of the mitogen-inducible isoform of cyclooxygenase (PGHS-2) in the skeletal myofibers compared to the interstitial fibroblasts. While the stretch-induced increase in cyclooxygenase enzymatic activity was not inhibited by tetrodotoxin and therefore was independent of cellular electrical activity, the G protein inhibitor pertussis toxin prevented stretch-induced cyclooxygenase activation. Pertussis toxin also inhibited stretch-induced increases in PGF_2α production, phospholipase D activation, and cell growth. It is concluded that stretch of skeletal muscle increases muscle cell growth through a G protein-dependent process involving the activation of cyclooxygenase, an immediate early gene product. © 1995 Wiley-Liss, Inc.     

Chronic exercise decreases cytokine production in healthy rat skeletal muscle

Skeletal muscle is the source of pro‐ and anti‐inflammatory cytokines, and recently, it has been recognized as an important source of interleukin‐6 (IL‐6). Acute physical exercise is known to induce a pro‐inflammatory cytokine profile in the plasma. However, the effect of chronic physical exercise in the production of pro‐ and anti‐inflammatory cytokines by the skeletal muscle has never been examined. We assessed IL‐6, TNF‐α, IL‐1β and IL‐10 levels in the skeletal muscle of rats submitted to endurance training. Animals were randomly assigned to either a sedentary group (S, n = 7) or an endurance exercise trained group (T, n = 8). Trained rats ran on a treadmill for 5 days week^?1 for 8 weeks (60% VO_2max). Detection of IL‐6, TNF‐α, IL‐1β and IL‐10 protein expression was carried out by ELISA. We found decreased expression of IL‐1β, IL‐6, TNF‐α and IL‐10 (28%, 27%, 32% and 37%, respectively, p < 0.05) in the extensor digital longus (EDL) from T, when compared with S. In the soleus, IL‐1β, TNF‐α and IL‐10 protein levels were similarly decreased (34%, 42% and 50%, respectively, p < 0.05) in T in relation to S, while IL‐6 expression was not affected by the training protocol. In conclusion, exercise training induced decreased cytokine protein expression in the skeletal muscle. These data show that in healthy rats, 8‐week moderate‐intensity aerobic training down regulates skeletal muscle production of cytokines involved in the onset, maintenance and regulation of inflammation, and that the response is heterogeneous according to fibre composition. Copyright © 2009 John Wiley & Sons, Ltd.     

Acetylcholinesterase activity in developing skeletal muscle cells

Acetylcholinesterase activity has been demonstrated biochemically and cytochemically in developing chick embryo skeletal muscle cells growing in culture. The enzyme shows the same pattern of drug sensitivity as that of adult skeletal muscle acetylcholinesterase and in present in cultured myogenic cells before the time of cell fusion, the formation of myotubes, and the subsequent increase in rate of myosin synthesis. Myogenic cell fusion is accompanied, however, by a large increase in activity of acetylcholinesterase. The enzyme activity is restricted in these cultures to myogenic cells. Neighboring fibroblasts show no cytochemical responses when challenged with techniques showing intense activity in myoblasts and myotubes. In addition, evidence is presented which strongly suggests that acetylcholinesterase activity in dividing myogenic cells is not constant over the cell cycle.     

Regulation of prostaglandin H synthase activity in dog urothelial cells.

TPA regulation of prostaglandin H synthase activity in primary and subcultured dog urothelial cells was investigated. Previous studies have demonstrated an early (0-2 hr) increase in PGE2 synthesis mediated by TPA which is dependent upon release of endogenous arachidonic acid by a phospholipase-mediated pathway. In this study, prostaglandin H synthase activity was assessed directly with microsomes and indirectly after addition of exogenous arachidonic acid at a maximum effective concentration (100 microM) to media. PGE2 synthesis, measured by radioimmunoassay, served as an index of prostaglandin H synthase activity. After a 24-hr incubation with 0.1 microM TPA or 1.0 microM A23187, arachidonic acid elicited significantly more PGE2 synthesis in agonist-treated cells than it did in control cells in primary culture. Microsomes from 24-hr TPA-treated cells exhibited significantly more prostaglandin H synthase activity than did those from control cells. In addition, the PGE2 content of overnight media was approximately 10-fold greater in TPA-treated cells than in control cells. The late (24 hr) response was more sensitive to lower concentrations of TPA than was the earlier (0-2 hr) response. TPA at 0.1 microM was a maximum effective dose for both responses. The 24-hr response was blocked by cycloheximide and staurosporine, inhibitors of protein synthesis and protein kinase C, respectively. Pretreatment of cells with aspirin, an irreversible inhibitor of prostaglandin H synthase, prior to addition of TPA did not prevent the late TPA-mediated increase in PGE2 synthesis. Subcultured cells exhibited both an early and a late TPA response. Only the early response was inhibited by aspirin pretreatment. Results suggest that the late response with TPA is caused by de novo synthesis of prostaglandin H synthase. Thus, primary and subcultured dog urothelial cells possess two distinct mechanisms for regulating signal transduction by arachidonic acid metabolism. This study provides a basis for assessing these mechanisms of signal transduction in urothelial cell lines and transformed cells.     

Mechanical strain increases gene transfer to skeletal muscle cells

Gene transfer techniques possess tremendous potential for treating diseases and for facilitating the study of basic physiological processes. However, further development of efficient and safe methods for gene transfer is needed. The purpose of this study was to test the hypothesis that mechanical strain increases the transfer of DNA to differentiated skeletal muscle cells. We tested this hypothesis by applying cyclic strain to cultured skeletal myotubes either prior to or immediately after the introduction of exogenous DNA complexed with lipids, with strains of varying magnitude (10%, 20% and 30%), number (1800, 3600 and 7200 strain cycles) and frequency (0.5, 1.0 and 1.5 Hz). Results demonstrated that DNA transfection was increased by exposing muscle cells to cyclic strain, and that strain magnitude, number and frequency each influenced DNA transfection. Optimal strain conditions (20% strain magnitude, 3600 cycles applied at 1 Hz) were utilized to examine the role of membrane transport systems in strain-induced increases in DNA transfection. Filipin III was used to inhibit caveolar transport and was found to inhibit strain-mediated increases in DNA transfection, whereas chlorpromazine, used to inhibit clathrin-coated vesicle transport, had no effect. These results indicate that mechanical strain may be an effective method for increasing DNA transfection in skeletal muscle through enhanced caveolar transport.     

Mechanical stimulation of skeletal muscle generates lipid-related second messengers by phospholipase activation

Repetitive mechanical stimulation of cultured avian skeletal muscle increases the synthesis of prostaglandins (PG) E₂ and F_2α which regulate protein turnover rates and muscle cell growth. These stretch-induced PG increases are reduced in low extracellular calcium medium and by specific phospholipase inhibitors. Mechanical stimulation increases the breakdown rate of ³H-arachidonic acid labelled phospholipids, releasing free ³H-arachidonic acid, the rate-limiting precursor of PG synthesis. Mechanical stimulation also increases ³H-arachidonic acid labelled diacylglycerol formation and intracellular levels of inositol phosphates from myo-[2-³H]inositol labelled phospholipids. Phospholipase A₂ (PLA₂), phosphatidylinositol-specific phospholipase C (PLC), and phospholipase D (PLD) are all activated by stretch. The stretch-induced increases in PG production, ³H-arachidonic acid labelled phospholipid breakdown, and ³H-arachidonic acid labelled diacylglycerol formation occur independently of cellular electrical activity (tetrodotoxin insensitve) whereas the formation of inositol phosphates from myo-[2-³H]inositol labelled phospholipids is dependent on cellular electrical activity. These results indicate that mechanical stimulation increases the lipid-related second messengers arachidonic acid, diacylglycerol, and PG through activation of specific phospholipases such as PLA₂ and PLD, but not by activation of phosphatidylinositol-specific PLC. © 1993 Wiley-Liss, Inc.     

Regulation of glycogen concentration and glycogen synthase activity in skeletal muscle of insulin-resistant rats

The aim of this study was to investigate the effect of insulin resistance on glycogen concentration and glycogen synthase activity in the red and white gastrocnemius muscles and to determine whether the inverse relationship existing between glycogen concentration and enzyme activity is maintained in insulin resistant state. These questions were addressed using 3 models that induce various degrees of insulin resistance: sucrose feeding, dexamethasone administration, and a combination of both treatments (dex+sucrose). Sucrose feeding raised triglyceride levels without affecting plasma glucose or insulin concentrations whereas dexamethasone and dex+sucrose provoked severe hyperinsulinemia, hyperglycemia and hypertriglyceridemia. Sucrose feeding did not alter muscle glycogen concentration but provoked a small reduction in the glycogen synthase activity ratio (-/+ glucose-6-phosphate) in red but not in white gastrocnemius. Dexamethasone administration augmented glycogen concentration and reduced glycogen synthase activity ratio in both muscle fiber types. In contrast, dex+sucrose animals showed decreased muscle glycogen concentration compared to dexamethasone group, leading to levels similar to those of control animals. This was associated with lower glycogen synthase activity compared to control animals leading to levels comparable to those of dexamethasone-treated animals. Thus, in dex+sucrose animals, the inverse relationship observed between glycogen levels and glycogen synthase activity was not maintained, suggesting that factors other than the glycogen concentration modulate the enzyme's activity. In conclusion, while insulin resistance was associated with a reduced glycogen synthase activity ratio, we found no correlation between muscle glycogen concentration and insulin resistance. Furthermore, our results suggest that sucrose treatment may modulate dexamethasone action in skeletal muscle.     

Mechanical loading regulates NOS expression and activity in developing and adult skeletal muscle

The hypothesis that changes in muscle activation and loadingregulate the expression and activity of neuronal nitric oxide (NO)synthase (nNOS) was tested using in vitro and in vivo approaches. Removal of weight bearing from rat hindlimb muscles for 10 days resulted in a significant decrease in nNOS protein and mRNAconcentration in soleus muscles, which returned to controlconcentrations after return to weight bearing. Similarly, theconcentration of nNOS in cultured myotubes increased by application ofcyclic loading for 2 days. NO release from excised soleus muscles wasincreased significantly by a single passive stretch of 20% or bysubmaximal activation at 2 Hz, although the increases were not additivewhen both stimuli were applied simultaneously. Increased NO release resulting from passive stretch or activation was dependent on thepresence of extracellular calcium. Cyclic loading of cultured myotubesalso resulted in a significant increase in NO release. Together, thesefindings show that activity of muscle influences NO production in theshort term, by regulating NOS activity, and in the long term, byregulating nNOS expression.

     

Induction of neuronal type nitric oxide synthase in skeletal muscle by chronic electrical stimulation in vivo

Reiser, Peter J., William O. Kline, and Pal L. Vaghy.Induction of neuronal type nitric oxide synthase in skeletal muscle by chronic electrical stimulation in vivo. J. Appl. Physiol. 82(4): 1250-1255, 1997.Fast-twitch skeletal muscles contain more neuronal-type nitricoxide synthase (nNOS) than slow-twitch muscles because nNOS is presentonly in fast (type II) muscle fibers. Chronic in vivo electricalstimulation of tibialis anterior and extensor digitorum longus musclesof rabbits was used as a method of inducing fast-to-slow fiber typetransformation. We have studied whether an increase in musclecontractile activity induced by electrical stimulation alters nNOSexpression, and if so, whether the nNOS expression decreases to thelevels present in slow muscles. Changes in the expression of myosinheavy chain isoforms and maximum velocity of shortening of skinnedfibers indicated characteristic fast-to-slow fiber type transformationafter 3 wk of stimulation. At the same time, activity of NOS doubled inthe stimulated muscles, and this correlated with an increase in theexpression of nNOS shown by immunoblot analysis. These data suggestthat nNOS expression in skeletal muscle is regulated by muscle activityand that this regulation does not necessarily follow the fast-twitchand slow-twitch pattern during the dynamic phase of phenotypetransformation.

     

Epidermal growth factor stimulation of prostaglandin E2 biosynthesis in amnion cells. Induction of prostaglandin H2 synthase

Amnion is believed to be a tissue of signal importance, anatomically and functionally, in the maintenance of pregnancy and during the initiation of parturition. Epidermal growth factor (EGF)-like agents cause a striking increase in the secretion of prostaglandin E2 (PGE2) in human amnion cells but only if arachidonic acid is present in the culture medium. To investigate the regulation of arachidonic acid metabolism by EGF-like agents in amnion, we used mEGF and human amnion cells in primary monolayer culture as a model system. The amount of PGE2 secreted into the culture medium was quantified by radioimmunoassay and the rate of conversion of [14C]arachidonic acid to [14C]PGE2 (PGH2 synthase activity) in cell sonicates was determined under optimal in vitro conditions. Treatment of amnion cells with mEGF led to a marked increase in the rate of production of PGE2. The specific activity of PGH2 synthase (viz. the combined activities of prostaglandin endoperoxide (PGH2) synthase and PGH2-PGE isomerase) was increased by 2-5-fold in cells treated with mEGF. Treatment of amnion cells with mEGF for 4 h did not affect the specific activities of phospholipase A2 or phosphatidylinositol-specific phospholipase C. By immunoisolation of newly synthesized, [35S]methionine-labeled PGH2 synthase, we found that mEGF stimulated de novo synthesis of the enzyme. Thus, mEGF acts in human amnion cells in primary monolayer culture to increase the rate of PGE2 biosynthesis by a mechanism that involves induction of PGH2 synthase; the manifestation of EGF action on PGE2 biosynthesis is dependent on the presence of nonesterified arachidonic acid.     

Regulation of calcineurin activity in insulin-secreting cells: stimulation by Hsp90 during glucocorticoid-induced apoptosis

Previously, we described that apoptotic cell death induced by the synthetic glucocorticoid dexamethasone (dex) is inhibited by calcineurin inhibitors, FK506 and deltamethrin, in insulin-secreting cells. The aim of the present study was to examine the mechanism of dex-dependent activation of calcineurin. In INS-1 cells cultured up to 4d with dex (100 nmol/l), the percentage of apoptosis, quantified by condensed nuclei and TUNEL positive cells, increased from 1% to 10.9%. FK506 inhibited dex-mediated cell death. Apoptosis was significantly higher at glucose concentrations that induce [Ca(2+)](i) oscillations than at low, non-stimulatory glucose. Dex had no acute effect on [Ca(2+)](i). Calcineurin activity, measured in control and dex-treated cell homogenates, revealed that maximal activity and the sensitivity to the substrate RII peptide was unaltered. However, dex treatment significantly increased enzyme activity at submaximal, physiological Ca(2+) concentrations. Dex did not stimulate the Ca(2+)-dependent protease calpain, known to activate calcineurin by cleavage, as no cleaved calcineurin was detectable. Furthermore, the calpain inhibitor ALLN did not counteract dex-dependent cell death. Western blotting revealed that in dex-treated cells heat shock protein 90 (Hsp90), a component of the glucocorticoid receptor (GR) known to stimulate calcineurin, was increased while calcineurin protein levels were unchanged. In immunoprecipitates with calcineurin antibodies, Hsp90 was only detected in dex-treated cell homogenates. These data suggest that dex-induced apoptosis involves release of Hsp90 from the stimulated GR complex, subsequent binding to and activation of calcineurin, that may contribute to dex-mediated cell death in the presence of high glucose.     

Glucose transport rate and glycogen synthase activity both limit skeletal muscle glycogen accumulation

We varied rates of glucose transport and glycogen synthase I (GS-I) activity (%GS-I) in isolated rat epitrochlearis muscle to examine the role of each process in determining the rate of glycogen accumulation. %GS-I was maintained at or above the fasting basal range during 3 h of incubation with 36 mM glucose and 60 microU/ml insulin. Lithium (2 mM LiCl) added to insulin increased glucose transport rate and muscle glycogen content compared with insulin alone. The glycogen synthase kinase-3beta inhibitor GF-109203 x (GF; 10 microM) maintained %GS-I about twofold higher than insulin with or without lithium but did not increase glycogen accumulation. When %GS-I was lowered below the fasting range by prolonged incubation with 36 mM glucose and 2 mU/ml insulin, raising rates of glucose transport with bpV(phen) or of %GS-I with GF produced additive increases in glycogen concentration. Phosphorylase activity was unaffected by GF or bpV(phen). In muscles of fed animals, %GS-I was approximately 30% lower than in those of fasted rats, and insulin-stimulated glycogen accumulation did not occur unless %GS-I was raised with GF. We conclude that the rate of glucose transport is rate limiting for glycogen accumulation unless %GS-I is below the fasting range, in which case both glucose transport rate and GS activity can limit glycogen accumulation.