Bonding to CAD-CAM Composites: An Interfacial Fracture Toughness Approach.

The objective of this study was to evaluate the interfacial fracture toughness (IFT) of composite cement with dispersed filler (DF) versus polymer-infiltrated ceramic network (PICN) computer-aided design and computer-aided manufacturing (CAD-CAM) composite blocks after 2 different surface pretreatments using the notchless triangular prism (NTP) test. Two DFs (Cerasmart [CRT] and Lava Ultimate [LVA]), 2 PICNs (Enamic [ENA] and experimental PICN [EXP]), and e.max CAD lithium disilicate glass-ceramic (EMX, control) prism samples were bonded to their counterparts with Variolink Esthetic DC composite cement after either hydrofluoric acid etching (HF) or gritblasting (GR). Both procedures were followed by silanization. All samples ( n = 30 per group) were thermocycled (10,000 cycles) and tested for their IFT in a water bath at 36°C. Moreover, representative samples from each group were subjected to a developed interfacial area ratio (Sdr) measurement by profilometry and scanning electron microscopy (SEM) characterization. EXP-HF gave the highest IFT (1.85 ± 0.39 MPa·m1/2), followed by EMX-HF and ENA-HF, while CRT-HF gave the lowest (0.15 ± 0.22 MPa·m1/2). PICNs gave significantly better results with HF, and DF showed better results with GR. A 2-way analysis of variance indicated that there were significantly higher IFT and Sdr for PICNs than for DF. A positive correlation ( r² = 0.872) was found between IFT and Sdr. SEM characterization showed the specific microstructure of the surface of etched PICNs, indicating the presence of a retentive polymer-based honeycomb structure. Etching of the typical double-network microstructure of PICNs causes an important increase in the Sdr and IFT, while DF should be gritblasted. DF exhibited significantly lower Sdr and IFT values than PICNs. The present results show the important influence of the material class and surface texture, and consequently the micromechanical bond, on the adhesive interface performance of CAD-CAM composites.

Neural progenitor fate decision defects, cortical hypoplasia and behavioral impairment in Celsr1-deficient mice.

The development of the cerebral cortex is a tightly regulated process that relies on exquisitely coordinated actions of intrinsic and extrinsic cues. Here, we show that the communication between forebrain meninges and apical neural progenitor cells (aNPC) is essential to cortical development, and that the basal compartment of aNPC is key to this communication process. We found that Celsr1, a cadherin of the adhesion G protein coupled receptor family, controls branching of aNPC basal processes abutting the meninges and thereby regulates retinoic acid (RA)-dependent neurogenesis. Loss-of-function of Celsr1 results in a decreased number of endfeet, modifies RA-dependent transcriptional activity and biases aNPC commitment toward self-renewal at the expense of basal progenitor and neuron production. The mutant cortex has a reduced number of neurons, and Celsr1 mutant mice exhibit microcephaly and behavioral abnormalities. Our results uncover an important role for Celsr1 protein and for the basal compartment of neural progenitor cells in fate decision during the development of the cerebral cortex.

Contribution of plasma membrane lipid domains to red blood cell (re)shaping.

Although lipid domains have been evidenced in several living cell plasma membranes, their roles remain largely unclear. We here investigated whether they could contribute to function-associated cell (re)shaping. To address this question, we used erythrocytes as cellular model since they (i) exhibit a specific biconcave shape, allowing for reversible deformation in blood circulation, which is lost by membrane vesiculation upon aging; and (ii) display at their outer plasma membrane leaflet two types of submicrometric domains differently enriched in cholesterol and sphingomyelin. We here reveal the specific association of cholesterol- and sphingomyelin-enriched domains with distinct curvature areas of the erythrocyte biconcave membrane. Upon erythrocyte deformation, cholesterol-enriched domains gathered in high curvature areas. In contrast, sphingomyelin-enriched domains increased in abundance upon calcium efflux during shape restoration. Upon erythrocyte storage at 4 °C (to mimick aging), lipid domains appeared as specific vesiculation sites. Altogether, our data indicate that lipid domains could contribute to erythrocyte function-associated (re)shaping.

P2Y2 receptor activation inhibits the expression of the sodium-chloride cotransporter NCC in distal convoluted tubule cells.

Luminal nucleotide stimulation is known to reduce Na(+) transport in the distal nephron. Previous studies suggest that this mechanism may involve the thiazide-sensitive Na(+)-Cl(-) cotransporter (NCC), which plays an essential role in NaCl reabsorption in the cells lining the distal convoluted tubule (DCT). Here we show that stimulation of mouse DCT (mDCT) cells with ATP or UTP promoted Ca(2+) transients and decreased the expression of NCC at both mRNA and protein levels. Specific siRNA-mediated silencing of P2Y2 receptors almost completely abolished ATP/UTP-induced Ca(2+) transients and significantly reduced ATP/UTP-induced decrease of NCC expression. To test whether local variations in the intracellular Ca(2+) concentration ([Ca(2+)]i) may control NCC transcription, we overexpressed the Ca(2+)-binding protein parvalbumin selectively in the cytosol or in the nucleus of mDCT cells. The decrease in NCC mRNA upon nucleotide stimulation was abolished in cells overexpressing cytosolic PV but not in cells overexpressing either a nuclear-targeted PV or a mutated PV unable to bind Ca(2+). Using a firefly luciferase reporter gene strategy, we observed that the activity of NCC promoter region from -1 to -2,200 bp was not regulated by changes in [Ca(2+)]i. In contrast, high cytosolic calcium level induced instability of NCC mRNA. We conclude that in mDCT cells: (1) P2Y2 receptor is essential for the intracellular Ca(2+) signaling induced by ATP/UTP stimulation; (2) P2Y2-mediated increase of cytoplasmic Ca(2+) concentration down-regulates the expression of NCC; (3) the decrease of NCC expression occurs, at least in part, via destabilization of its mRNA.

Ophiobolin A induces paraptosis-like cell death in human glioblastoma cells by decreasing BKCa channel activity.

Glioblastoma multiforme (GBM) is the most lethal and common malignant human brain tumor. The intrinsic resistance of highly invasive GBM cells to radiation- and chemotherapy-induced apoptosis accounts for the generally dismal treatment outcomes. This study investigated ophiobolin A (OP-A), a fungal metabolite from Bipolaris species, for its promising anticancer activity against human GBM cells exhibiting varying degrees of resistance to proapoptotic stimuli. We found that OP-A induced marked changes in the dynamic organization of the F-actin cytoskeleton, and inhibited the proliferation and migration of GBM cells, likely by inhibiting big conductance Ca(2+)-activated K(+) channel (BKCa) channel activity. Moreover, our results indicated that OP-A induced paraptosis-like cell death in GBM cells, which correlated with the vacuolization, possibly brought about by the swelling and fusion of mitochondria and/or the endoplasmic reticulum (ER). In addition, the OP-A-induced cell death did not involve the activation of caspases. We also showed that the expression of BKCa channels colocalized with these two organelles (mitochondria and ER) was affected in this programmed cell death pathway. Thus, this study reveals a novel mechanism of action associated with the anticancer effects of OP-A, which involves the induction of paraptosis through the disruption of internal potassium ion homeostasis. Our findings offer a promising therapeutic strategy to overcome the intrinsic resistance of GBM cells to proapoptotic stimuli.

A novel renal carbonic anhydrase type III plays a role in proximal tubule dysfunction.

Dysfunction of the proximal tubule (PT) is associated with variable degrees of solute wasting and low-molecular-weight proteinuria. We measured metabolic consequences and adaptation mechanisms in a model of inherited PT disorders using PT cells of ClC-5-deficient (Clcn5Y/-) mice, a well-established model of Dent's disease. Compared to cells taken from control mice, those from the mutant mice had increased expression of markers of proliferation (Ki67, proliferative cell nuclear antigen (PCNA), and cyclin E) and oxidative scavengers (superoxide dismutase I and thioredoxin). Transcriptome and protein analyses showed fourfold induction of type III carbonic anhydrase in a kidney-specific manner in the knockout mice located in scattered PT cells. Kidney-specific carbonic anhydrase type III (CAIII) upregulation was confirmed in other mice lacking the multiligand receptor megalin and in a patient with Dent's disease due to an inactivating CLCN5 mutation. The type III enzyme was specifically detected in the urine of mice lacking ClC-5 or megalin, patients with Dent's disease, and in PT cell lines exposed to oxidative stress. Our study shows that lack of PT ClC-5 in mice and men is associated with CAIII induction, increased cell proliferation, and oxidative stress.

In situ measurements of calpain activity in isolated muscle fibres from normal and dystrophin-lacking mdx mice.

Calpains are Ca(2+)-activated proteases that are thought to be involved in muscle degenerative diseases such as Duchenne muscular dystrophy. Status and activity of calpains in adult muscle fibres are poorly documented. We report here in situ measurements of calpain activity in collagenase-isolated fibres from C57 mice and form two models of dystrophy: dystrophin-deficient mdx and calpain-3 knocked-out mice. Calpain activity was measured using a permeant, fluorogenic substrate and its Ca(2+) dependence was studied. A 30-fold change of activity was observed between the lowest and the highest steady-state Ca(2+) availability. Fast transient changes of [Ca(2+)](i) induced by electrical stimulation or KCl-dependent depolarization were ineffective in activating calpain. Slow [Ca(2+)] transients, as elicited during depletion of Ca(2+) stores, Ca(2+) store repletion and hypo-osmotic swelling were able to activate calpain. On return to resting conditions, calpain activity recovered its basal rate within 10 min. In resting intact muscle, mu-calpain was predominantly in the 80 kDa native form, with a small fraction in the 78 kDa autolysed form. The latter is thought to be responsible for the activity measured in our conditions. Calpain activity in mdx fibres showed an average 1.5-fold increase compared to activity in C57 fibres. This activity was reduced by a 10-fold lowering of [Ca(2+)](o). Calpain-3-deficient fibres showed about the same increase, thus calpain-3 did not contribute to the activity measured here and calpain activation is not specific to dystrophin deficiency. In fibres from transgenic mice over-expressing calpastatin, a 40-50% reduction of calpain activity was observed, as with synthetic drugs (Z-Leu-Leu-CHO and SNT198438). We provide novel information on the physiological factors that control calpain activity in situ, particularly the effect of intracellular Ca(2+) transients that occur in excitation-contraction coupling, Ca(2+) store depletion and refilling, and activation of mechanosensitive Ca(2+) channels.

Regulation of store-operated calcium entries and mitochondrial uptake by minidystrophin expression in cultured myotubes.

Defective expression of dystrophin in muscle cells is the primary feature of Duchenne muscular dystrophy (DMD), which is accompanied by fiber necrosis and intracellular calcium mishandling. These features led to the hypothesis that dystrophin could control calcium movements. Calcium mishandling in human DMD myotubes is dependent on contraction and/or calcium release activity, suggesting the involvement of channels being activated during these processes. Forced expression of minidystrophin at the plasma membrane of dystrophin-deficient Sol8 myotubes reactivates appropriate sarcolemmal expression of dystrophin-associated proteins and results in normal calcium homeostasis. In active dystrophic myotubes, store-operated calcium channels could be responsible for a sustained calcium influx in muscle cells. We show here that depletion of calcium stores (sarcoplasmic reticulum) by repetitive activation of calcium release and blockade of SERCA leads to a calcium influx. In myotubes expressing recombinant minidystrophin, these store-dependent influxes were reduced to a level similar to that observed in myotubes expressing native dystrophin. High store-dependent calcium influxes in dystrophin-deficient myotubes were associated with sustained cytosolic calcium transients and high intramitochondrial entries, while lower store-dependent calcium influx in myotubes expressing minidystrophin resulted in shorter calcium transients and reduced calcium uptake into mitochondria. We propose that minidystrophin negatively regulates sarcolemmal store-dependent calcium channels, which reduces store-dependent calcium influx, as well as its mitochondrial uptake. Forced expression of minidystrophin in dystrophic cells might restore the regulation of sarcolemmal store-dependent channels, which could protect against calcium mishandling.

Consequence of parvalbumin deficiency in the mdx mouse: histological, biochemical and mechanical phenotype of a new double mutant.

We tested the hypothesis whether the mild dystrophy in mdx mice could result from the contribution of the cytosolic calcium buffer parvalbumin in maintaining a normal cytosolic [Ca2+]i, in spite of an increased passive Ca2+ influx. By crossing mdx mice with parvalbumin-deficient mice, double mutant mice, lacking both dystrophin and parvalbumin, were obtained. Though resting cytosolic [Ca2+]i and total calcium content were similar to that of mdx muscles, this new animal model presented a slightly more severe phenotype than the mdx mouse. Muscle pseudo-hypertrophy, the density of myotubes and of centronucleated fibres as well as the loss of IIB fibres were all increased in parvalbumin-deficient mdx mice. Many of these deficits were overcome in late adulthood, albeit fibrosis was clearly more pronounced than in mdx muscles. At 90 days, parvalbumin-deficient mdx mice showed higher levels of creatine phosphokinase and lower muscle strength, in vivo, than mdx mice. Isometric tension of isolated muscle was reduced, but the susceptibility to eccentric contraction was not increased. The slight aggravation of muscle dystrophy observed in mdx mice deprived of parvalbumin cannot explain the severity of the affection observed in xmd dogs and Duchenne dystrophy patients where parvalbumin is constitutively not expressed.

New aspects of calcium signaling in skeletal muscle cells: implications in Duchenne muscular dystrophy.

Calcium is the most ubiquitous second messenger. Its concentration inside the cell is tightly regulated by a series of mechanisms, among which some have been extensively studied in nonmuscle cells. This is the case of the "store-operated entry of Ca(2+)", the uptake of Ca(2+) by mitochondria and the inositol 1,4,5-trisphosphate (IP(3)) cascade. These processes were recently found to be also present in skeletal muscle and are reviewed here. The "store-operated entry of Ca(2+)" allows the refilling of the stores after muscle fiber depolarization and is activated even after a partial depletion of the sarcoplasmic reticulum (SR). The uptake of Ca(2+) by mitochondria accelerates muscle relaxation and allows the adaptation of ATP supply to the increased energy demand. IP(3) receptors are found in the nuclear envelope and are involved in Ca(2+) waves propagating from one nucleus to another. This pathway is possibly involved in gene expression regulation. Finally, cytosolic Ca(2+) buffers like parvalbumins modify [Ca(2+)](i) transients and, therefore, muscle mechanics. The importance of these regulation mechanisms is also evaluated in Duchenne muscular dystrophy (DMD), a disease in which impairment of [Ca(2+)](i) homeostasis has been postulated but remains, however, controversial. This genetic disease is indeed characterized by the absence of a cytoskeletal protein called dystrophin, a situation leading to a disorganization of the cytoskeleton and to an abnormal influx of Ca(2+). How this increased entry of Ca(2+) affects the local concentration of Ca(2+) in subcellular compartments and whether this process is involved in the development of the disease are still unclear.

Long-term study of Ca(2+) homeostasis and of survival in collagenase-isolated muscle fibres from normal and mdx mice.

Skeletal muscles of the mdx mouse lack dystrophin offering the possibility to study the role of intracellular Ca(2+) ions in fibre degeneration. Flexor digitorum brevis muscles of 3-month-old mdx and normal mice were dissociated with collagenase; fibres were maintained in culture for 6 days (d0 to d5) and their survival was assessed. Cytosolic [Ca(2+)], passive Mn(2+) influx (indicative of Ca(2+) influx) and activity of mechanosensitive/voltage-independent Ca(2+) channels were studied over the same period. Survival of normal fibres declined steadily from d0 to d3, but an acceleration of fibre death occurred in mdx fibres from d1 to d2. This could be greatly reduced but not abolished by lowering external [Ca(2+)] 10-fold. In the d0-d5 period, both mdx and normal fibres showed transient increases of Mn(2+) influx and activity of the Ca(2+) channels; these peaked at d1 and disappeared by d3-d4. Increases were always significantly larger in mdx fibres. Altogether, over the 6 days, 130 paired measurements of [Ca(2+)](i) and Mn(2+) influx were made on 68 fibres from mdx and 62 fibres from normal mice. In 90 % of the fibres, [Ca(2+)](i) remained within the 25-85 nM limits while Mn(2+) influx varied more than 10-fold. The median for Mn(2+) influx was 45 % greater in fibres from mdx mice than in fibres from control C57 mice. However, there was no significant difference between [Ca(2+)](i) medians in fibres from normal and mdx mice. Addition of 25-75 nM of a Ca(2+) ionophore (4-bromo-A23187) to the medium did not affect the level of cytosolic [Ca(2+)] in both types of fibres, while markedly increasing the rate of Mn(2+) influx, as expected. Thus, Ca(2+) homeostasis was equally robust in mdx and normal fibres. The remaining 10 % of the fibres showed, at d1, high levels of Mn(2+) influx and/or elevated [Ca(2+)](i) above 100 nM. This did not affect survival of normal fibres but was probably responsible of the increased death rate in mdx fibres.

Intracellular pH regulation in isolated fast-twitch skeletal muscle from dystrophin-deficient mouse.

In muscles from anaesthetized dystrophin-deficient mdx mice, exercise results in a stronger acidification and a slower intracellular pH recovery compared to control mice. We examined whether this observation could be attributed to defective H+-carriers in dystrophin-lacking muscles. Immunohistochemistry and Western blots revealed no defect in mdx muscles for the presence of the lactate-/H+co-transporter MCT4 and of the Na+/H+ antiporter NHE1, the main H+-carriers active in fast-twitch skeletal muscle after exercise. Functional tests of the H+-transporters, on isolated muscles submitted to identical flow of superfusion, were performed in conditions meant to lower intracellular pH: repetitive electrical stimulation or NH4Cl pre-pulse. These revealed no defect in intracellular pH recovery in mdx muscles. Therefore, we conclude that impaired intracellular pH regulation in anaesthetized mdx mice is not attributable to a reduced presence or activity of H+-extruders. We propose that CO2 washout might be slowed down in vivo in mdx muscles because of the defective vascular response in contracting muscles from these mice.

Involvement of trp-2 protein in store-operated influx of calcium in fibroblasts.

Mammalian homologues of the Drosophila melanogaster transient receptor potential (trp) gene have been proposed to encode store-operated channels. This assertion essentially stays on the fact that expression of different trp proteins produces trans-membrane cation currents. However, the selectivity of the expressed channels and their mode of activation, in particular, their dependence to store depletion appears to be quite variable. In the present work, we adopted an anti-sense strategy to study this question in transfected Chinese hamster ovary cells expressing the rat neurotensin receptor (CHO-NTR cells), a cellular model characterized by its very large store-dependent entry of Ca(2+). We identified different trp transcripts by RT-PCR, the trp-1 and trp-2 transcripts being by far the most abundant. CHO-NTR cells were then transfected with a mouse trp-2 anti-sense construct (CHO-NTR-TRP2AS cells). We showed that in these cells, trp-2 mRNA was suppressed in comparison with cells transfected with a control plasmid. The store-operated entry of Ca(2+) was evaluated after store depletion by an IP(3)-dependent mechanism (neurotensin stimulation) or by direct inhibition of the endoplasmic reticulum Ca(2+)ATPase (thapsigargin stimulation). In both cases, store-dependent entry of Ca(2+) was largely reduced in CHO-NTR-TRP2AS cells in comparison with control cells, suggesting that trp-2 protein might constitute a functional subunit of store-operated channels.

Evidence for the dual coupling of the rat neurotensin receptor with pertussis toxin-sensitive and insensitive G-proteins.

We previously demonstrated the functional coupling of the rat neurotensin receptor NTS1 with G-proteins on transfected CHO cell homogenates by showing modulation of agonist affinity by guanylyl nucleotides and agonist-mediated stimulation of [(35)S]GTP gamma S binding. In the present study, we observed that G(i/o)-type G-protein inactivation by pertussis toxin (PTx) resulted in a dramatic reduction of the NT-induced [(35)S]GTP gamma S binding whereas the effect of guanylyl nucleotide was almost not affected. As expected, NT-mediated phosphoinositide hydrolysis and intracellular calcium mobilization were not altered after PTx treatment. This suggests the existence of multiple signaling cascades activated by NT. Accordingly, using PTx and the PLC inhibitor U-73122, we showed that both signaling pathways contribute to the NT-mediated production of arachidonic acid. These results support evidence for a dual coupling of the NTS1 with PTx-sensitive and insensitive G-proteins.

Tetanus relaxation of fast skeletal muscles of the mouse made parvalbumin deficient by gene inactivation.

The effects of tetanus duration on the relaxation rate of extensor digitorum longus (EDL) and flexor digitorum brevis (FDB) muscles were studied in normal (wild-type, WT) and parvalbumin-deficient (PVKO) mice, at 20 C. In EDL of PVKO, the relaxation rate was low and unaffected by tetanus duration (< 3.2 s). In contrast, the relaxation rate of WT muscles decreased when tetanus duration increased from 0.2 to 3.2 s. In WT muscles, fast relaxation recovered as the rest interval increased. Specific effect of parvalbumin was asserted by calculating the difference in relaxation rate between WT and PVKO muscles. For EDL, the rate constant of relaxation slowing was 1.10 s-1 of tetanization; the rate constant of relaxation recovery was 0.05 s-1 of rest. In FDB, the effects of tetanus duration on WT and PVKO muscles were qualitatively similar to those observed in EDL. Relaxation slowing as tetanus duration increases, reflects the progressive saturation of parvalbumin by Ca2+, while recovery as rest interval increases reflects the return to Ca2+-free parvalbumin. At all tetanus durations, relaxation rate still remained slightly faster in WT muscles. This suggested that parvalbumin facilitates calcium traffic from myofibrils to the SR. No difference was found between WT and PVKO muscles for: (i) the expression of the fast isoforms of myosin heavy chains, (ii) the force-velocity relationship and maximal shortening velocity and (iii) the Ca2+-activated ATPase activity from isolated preparations of the sarcoplasmic reticulum (SR).

Effect of adrenaline on the post-tetanic potentiation in mouse skeletal muscle.

We report the influence of adrenergic stimulation on the amplitude and time course of post-tetanic potentiation of twitch contraction. This was complemented by measurements of the peak of [Ca2+]i transients in twitches and of the level of myosin light chain 2 (LC2) phosphorylation, before, 20 and 300 s after the conditioning tetanus. Soon after the tetanus, twitch potentiation and increases of LC2 phosphorylation and of [Ca2+]i peak were similar in control conditions and in the presence of adrenaline. In control conditions, twitch potentiation, LC2 phosphorylation and [Ca2+]i peak returned to, or close to, pre-tetanic values in 300 s. On the contrary, in the presence of adrenaline, twitch potentiation and LC2 phosphorylation were partially or fully maintained respectively, while the increase of [Ca2+]i peak was not. This situation allowed us to analyse the relative contributions of elevated LC2 phosphorylation and [Ca2+]i peak in the twitch post-tetanic potentiation phenomenon. Moreover, it was shown that the increase of LC2 phosphorylation (up to 0.5 mol P/mol LC2) affected neither the kinetic parameters of the twitch nor the maximal velocity of shortening. It is proposed that the maintenance of LC2 phosphorylation in the presence of adrenaline results from the inhibition of myosin light chain phosphatase. This could be achieved through the production of the active, phosphorylated form of the inhibitor-1, an endogenous inhibitor, which binds to the catalytic sub-units common to class 1 protein phosphatases.

Effects of guandinoethane sulfonate on contraction of skeletal muscle.

Guanidinoethane sulfonic acid (GES), a chemical and biological analog of taurine, decreases rat muscle taurine content when added to drinking water. Over the same period, GES appears in muscle. GES supplementation is often used to study the effect of taurine depletion on physiological mechanisms, without taking into account the possible actions of GES. The purpose of the present study was to investigate the specific actions of GES on contraction of skeletal muscle. In mice EDL muscle, the time delay needed to observe a 20% force decrease after the end of a tetanic stimulation was higher in GES-supplemented than in control muscle. This observation in GES-supplemented muscle could be explained by the action of taurine or GES on several targets, beside others the rate of Ca2+ uptake by sarcoplasmic reticulum (SR) and the Ca2+ sensitivity of myofilaments. SR of rat EDL was isolated by successive centrifugations. The effect of 20 mM taurine or GES on the rate of Ca2+ uptake by SR was measured with the fluorescent Ca2+ indicator fura-2. The results show that the rate of Ca2+ uptake by SR is not modified in the presence of taurine or GES. The Ca2+ sensitivity of myofilaments was studied in chemically skinned fibers in the presence of 20 mM taurine or GES. Both taurine and GES increased the myofilament sensitivity to Ca2+. Thus, the prolonged relaxation time of GES-supplemented muscle can be attributed to an increase in myofilament sensitivity to Ca2+. This higher sensitivity is not due to a decrease in muscle taurine content but rather to an increased GES concentration.

Regulation of IGF-I, IGFBP-4 and IGFBP-5 gene expression by loading in mouse skeletal muscle.

Gene expression of IGF-I, IGFBP-4 and IGFBP-5 was studied in hindhimb skeletal muscle of mice, which were either overloaded or unloaded for 8 days. Overloading induced a 15% hypertrophy in soleus muscle associated with a 60% increase of IGF-I transcript levels and a doubling of IGFBP-4 mRNA levels. IGFBP-5 mRNA levels were decreased to one third of the control value. Changes in IGFBPs mRNA always preceded changes in IGF-I gene expression. Unloading by hindlimb suspension resulted in atrophy of soleus muscle (20%) and phenotype change towards the fast type associated with a transient decrease of IGF-I mRNA (30%) and a sustained increase (x2) of IGFBP-5 transcript. These alterations in IGFBPs expression, in unloaded or overloaded soleus, suggest that they may play a role in skeletal muscle adaptation to changes in loading.

Effects of nitric oxide on the contraction of skeletal muscle.

A review of the literature suggests that the effects of nitric oxide (NO) on skeletal muscles fibers can be classified in two groups. In the first, the effects of NO are direct, due to nitrosation or metal nitrosylation of target proteins: depression of isometric force, shortening velocity of loaded or unloaded contractions, glycolysis and mitochondrial respiration. The effect on calcium release channels varies, being inhibitory at low and stimulatory at high NO concentrations. The general consequence of the direct effects of NO is to 'brake' the contraction and its associated metabolism. In the second group, the effects of NO are mediated by cGMP: increase of the shortening velocity of loaded or unloaded contractions, maximal mechanical power, initial rate of force development, frequency of tetanic fusion, glucose uptake, glycolysis and mitochondrial respiration; decreases of half relaxation time of tetanus and twitch, twitch time-to-peak, force maintained during unfused tetanus and of stimulus-associated calcium release. There is negligible effect on maximal force of isometric twitch and tetanus. The general consequence of cGMP-mediated effects of NO is to improve mechanical and metabolic muscle power, similar to a transformation of slow-twitch to fast-twitch muscle, an effect that we may summarize as a 'slow-to-fast' shift.

Uptake and release of Ca2+ by the endoplasmic reticulum contribute to the oscillations of the cytosolic Ca2+ concentration triggered by Ca2+ influx in the electrically excitable pancreatic B-cell.

The role of intracellular Ca2+ pools in oscillations of the cytosolic Ca2+ concentration ([Ca2+]c) triggered by Ca2+ influx was investigated in mouse pancreatic B-cells. [Ca2+]c oscillations occurring spontaneously during glucose stimulation or repetitively induced by pulses of high K+ (in the presence of diazoxide) were characterized by a descending phase in two components. A rapid decrease in [Ca2+]c coincided with closure of voltage-dependent Ca2+ channels and was followed by a slower phase independent of Ca2+ influx. Blocking the SERCA pump with thapsigargin or cyclopiazonic acid accelerated the rising phase of [Ca2+]c oscillations and increased their amplitude, which suggests that the endoplasmic reticulum (ER) rapidly takes up Ca2+. It also suppressed the slow [Ca2+]c recovery phase, which indicates that this phase corresponds to the slow release of Ca2+ that was taken up by the ER during the upstroke of the [Ca2+]c transient. Glucose promoted the buffering capacity of the ER and amplified the slow [Ca2+]c recovery phase. The slow phase induced by high K+ pulses was not affected by modulators of Ca2+- or inositol 1,4,5-trisphosphate-induced Ca2+ release, did not involve a depolarization-induced Ca2+ release, and was also observed at the end of a rapid rise in [Ca2+]c triggered from caged Ca2+. It is attributed to passive leakage of Ca2+ from the ER. We suggest that the ER displays oscillations of the Ca2+ concentration ([Ca2+]ER) concomitant and parallel to [Ca2+]c. The observation that thapsigargin depolarizes the membrane of B-cells supports the proposal that the degree of Ca2+ filling of the ER modulates the membrane potential. Therefore, [Ca2+]ER oscillations occurring during glucose stimulation are likely to influence the bursting behavior of B-cells and eventually [Ca2+]c oscillations.