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Biomedical subjects

J M Gillis

Publications and source records attributed to J M Gillis.

At least 37 records · Page 2Linked to original sources
PubMed1997

Fructose-induced increase in intracellular free Mg2+ ion concentration in rat hepatocytes: relation with the enzymes of glycogen metabolism.

In rat hepatocytes subjected to a fructose load, ATP content decreased from 3.8 to 2.6 micromol/g of cells. Under these conditions, the intracellular free Mg2+ ion concentration,as measured with mag-fura 2, increased from 0.25 to 0.43 micromol/g of cells and 0.35 micromol of Mg2+ ions were released per g of cells in the extracellular medium. Therefore the increase in the intracellular free Mg2+ ion concentration was less than expected from the decrease in ATP, indicating that approx. 80% of the Mg2+ ions released from MgATP2- were buffered inside the cells. When this buffer capacity was challenged with an extra Mg2+ ion load by blocking the fructose-induced Mg2+ efflux, again approx. 80% of the extra Mg2+ ion load was buffered. The remaining 20% appearing as free Mg2+ions in fructose-treated hepatocytes could act as second messenger for enzymes having a Km for Mg2+ in the millimolar range. Fructose activated glycogen synthase and glycogen phosphorylase, although both the time course and the dose-dependence of activation were different. This was reflected in a stimulation of glycogen synthesis with concentrations of fructose below 5 mM. Indeed, activation of glycogen synthase reached a maximum at 30 min of incubation and was observed with small (5 mM or less) concentrations of fructose, whereas the activation of glycogen phosphorylase was almost immediate (within 5 min) and maximal with large doses of fructose. The fructose-induced activation of glycogen phosphorylase, but not that of glycogen synthase, could be related to an increase in free Mg2+ ion concentration.

Animals↗
PubMed1997

Inhibition of mitochondrial calcium uptake slows down relaxation in mitochondria-rich skeletal muscles.

Isolated fibres from various muscles were skinned mechanically in oil. From a Ca2+-loaded micropipette, local applications of Ca2+ were made. These produced a limited contraction which relaxed spontaneously. The time-course of sarcomere shortening and re-lengthening was recorded by microcinephotography. Application of Ruthenium Red, a potent and specific inhibitor of Ca2+ uptake by mitochondria, did not affect the contraction-relaxation cycles of typical glycolytic white fibres (frog sartorius, pigeon breast). By contrast, Ruthenium Red greatly slowed down the relaxation rate in mitochondria-rich fibres (rat soleus and rabbit masseter). In these fibres, Ca2+ uptake by mitochondria seems to play an active role in promoting relaxation.

Animals↗
PubMed1997

Polymerization of myosin on activation of rat anococcygeus smooth muscle.

The in vivo state of assembly of myosin in vertebrate smooth muscle is controversial. In vitro studies on purified smooth muscle myosin show that it is monomeric (10S) under relaxing conditions and filamentous under contraction conditions. Electron microscopic and antibody labelling studies of intact smooth muscles, on the other hand, suggest that myosin is filamentous in the relaxed as well as the contracting state and that 10S myosin occurs only in trace amounts. However, birefringence, conventional EM and X-ray diffraction evidence suggests that in certain smooth muscles in vivo (e.g. rat anococcygeus), while myosin filaments exist in the relaxed state, their number increases on contraction. Here, we have used low temperature electron microscopic techniques (rapid freezing followed by freeze-substitution), which preserve labile components in close to their in vivo state, to detect any change in filament number on contraction. The results from rat anococcygeus have been compared with those from guinea pig taenia coli, in which other techniques have revealed no change in filament number. In the anococcygeus, we find evidence for a 23% increase in filament density in transverse sections of contracting muscle compared with relaxed muscle. In the taenia coli we find no change. These results are in qualitative agreement with earlier findings. They provide evidence for polymerization of myosin in contracting rat anococcygeus, and suggest that this process is subtle and occurs only in some smooth muscles.

Animals↗
PubMed1997

Expression of truncated utrophin leads to major functional improvements in dystrophin-deficient muscles of mice.

Dystrophin-deficient mice (mdx) expressing a truncated (trc) utrophin transgene show amelioration of the dystrophic phenotype. Here we report a multifunctional study demonstrating that trcutrophin expression leads to major improvements of the mechanical performance of muscle (that is, force development, mechanical resistance to forced lengthenings and maximal spontaneous activity) and of the maintenance of the intracellular calcium homeostasis. These are two essential functions of muscle fibers, known to be impaired in mdx mouse muscles and Duchenne muscular dystrophy (DMD) patients. Our results bring strong support to the hypothesis that muscle wasting in dystrophin-deficient DMD patients could be prevented by upregulation of utrophin.

Animals↗
PubMed1996

Functional protection of dystrophic mouse (mdx) muscles after adenovirus-mediated transfer of a dystrophin minigene.

Fast skeletal muscles of mdx (X chromosome-linked muscular dystrophy) mice were injected after birth with a recombinant adenovirus containing a minidys- trophin gene, a 6.3-kbp cDNA coding for the N- and C-terminal ends of dystrophin. Adult muscles were challenged by forced lengthening during tetanic contractions. Stretch-induced mechanical and histological damages were much reduced in injected muscles, in direct proportion of the Miniber of fibers expressing minidystrophin. Damaged fibers were preferentially found among minidystrophin-negative regions. Minidystrostrophin confers an important functional and structural protection of limb muscles against high mechanical stress, even after a partial somatic gene transfer.

Adenoviridae↗
PubMed1996

Role of [Ca2+]i in "Ca2+ stores depletion-Ca2+ entry coupling' in fibroblasts expressing the rat neurotensin receptor.

1. Transfected Chinese hamster ovary fibroblasts expressing the rat neurotensin receptor were used to study the 'Ca2+ stores depletion-Ca2+ entry coupling' which follows stimulation with neurotensin and liberation of InsP3. 2. This coupling could be dissociated in time. Firstly, stores depletion was produced by neurotensin or thapsigargin which caused a first [Ca2+]i transient in a Ca(2+)-free external medium. Secondly, readmission of external Ca2+ produced an influx of Ca2+ and a second [Ca2+]i transient. 3. Various concentrations of thapsigargin (20 nM to 1 microM) were used to produce complete stores depletion with small or large first peaks of [Ca2+]i. Upon return to external Ca2+, small or large second [Ca2+]i peaks were observed. The amplitudes of both peaks were positively correlated. 4. The Ca2+ entry which followed stores depletion could occur at very low basal values of [Ca2+]i, was accelerated by okadaic acid and inhibited by staurosporine and the calmodulin antagonist W-7. 5. It is concluded that the rise in [Ca2+]i during Ca2+ stores depletion is an essential parameter which determines the size of the subsequent Ca2+ entry.

Animals↗
PubMed1996

Membrane abnormalities and Ca homeostasis in muscles of the mdx mouse, an animal model of the Duchenne muscular dystrophy: a review.

Muscles of the mdx mouse lack dystrophin, a cytoskeletal protein. Mdx fibres exhibit an increased fragility to hypo-osmotic shock and to forced lengthening, an abnormal opening time of stretch-sensitive calcium channels. The question of a chronic elevated [Ca2+]i value is a matter of controversy. We have analysed Ca homeostasis in smooth and skeletal muscles from the adult mdx mouse. The wall of the vas deferens was loaded with the fluorescent Ca indicator Fura-2-AM (cell-diffusible). Resting [Ca2+]i was measured after changes of the electrochemical potential for Ca2+ and after KCl or electrical stimulations. In no instance was a difference observed between these and similar muscles from control mice. Single striated fibres were isolated by collagenase treatment of the flexor digitorum brevis muscle and loaded with Fura-2-AM. The value of resting [Ca2+]i was measured using an in situ calibration procedure which took account of Ca buffering by Fura-2. A chronic increase of cytosolic Ca2+ was not confirmed. The expression of the intracellular Ca-binding protein, parvalbumin, was measured. It increased by about threefold in fast mdx muscles (tibialis anterior) but remained undetectable in the soleus. It is hypothesized that parvalbumin helps to maintain [Ca2+]i within normal values. This hypothesis will be discussed in connection with dystrophy phenotypes in mutant dogs and in human patients.

Animals↗
PubMed1995

Site-dependent pathological differences in smooth muscles and skeletal muscles of the adult mdx mouse.

This study presents a survey of the morphometric characteristics, the regeneration rate, and the extent of muscle dystrophy in several smooth and skeletal muscles from adult mdx mice, an animal model of the Duchenne muscular dystrophy (DMD). Smooth muscles from adult mdx mice showed neither cell necrosis nor fibrosis. As compared to control C57 mice, the thickness of the mdx smooth muscle was normal in the vascular and urogenital layers but significantly reduced in the digestive layers, a finding relevant to clinical reports of gastrointestinal dilatation in DMD patients, and suggesting that gastrointestinal dysfunctions should be systemically searched for in DMD patients. Adult mdx skeletal muscles, however, presented different patterns of muscle suffering: either absent (esophagus); very mild (trunk and limb muscles); or severe (diaphragm). In these three conditions we studied the fiber diameters, the nuclei locations, and the regeneration rate. From this comparative study, it seems that severe dystrophy occurs in muscle tissues showing large fiber diameter and peripheral location of the nuclei. We showed that this combination occurs in the mouse diaphragm which is thus a realistic model for human DMD muscles.

Animals↗
PubMed1995

Lack of rapid desensitization of Ca2+ responses in transfected CHO cells expressing the rat neurotensin receptor despite agonist-induced internalization.

Transfected Chinese hamster ovary cells were used as a model for the study of the desensitization of the neurotensin receptor at the second messenger level. Stimulation with nanomolar concentrations of neurotensin elicited rapid rises in the cytosolic calcium concentration ([Ca2+]i), which remained elevated throughout the peptide application. A significant response was already detected with neurotensin concentrations as low as 0.01 nM. This high efficiency of neurotensin in mediating this calcium response contrasts with the nanomolar affinity of the peptide for its receptor measured in binding experiments. Evidence indicated that the initial elevation of the [Ca2+]i resulted from release of Ca2+ from intracellular stores, whereas the sustained response involved an influx of extracellular origin. Return to the basal level was only reached after extensive washing of the peptide or its displacement with the neurotensin receptor antagonist SR48692. After washing, further stimulations were still able to mediate an increase in the [Ca2+]i, indicating an apparent absence of rapid desensitization of the intracellular signaling pathway that mediates calcium mobilization. In contrast with this absence of response desensitization, the neurotensin receptors were found to internalize after stimulation with the peptide. This internalization was maximal after 30 min and accounted for approximately 70% of the number of neurotensin binding sites located at the cell surface. These results indicate that despite the functional properties of the rat neurotensin receptor present in Chinese hamster ovary cells after transfection, the intracellular signaling pathway triggered by stimulation with neurotensin seems to be resistant to desensitization. This might be related to the high efficiency of the intracellular signaling pathway coupled to the neurotensin receptor observed in these cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

[Role of intracellular calcium in the programmed cell death of prostatic cancer cells].

The growth of any tissue depends on the quantitative relationship between the rate of cell proliferation and cell death. In normal adults tissues, this steady-state balance is regulated by a series of both systemic hormones and local growth factors. Programmed cell death, also called apoptosis, is one of the two pathways for cell death. In programmed cell death, specific intracellular signals induce the cell to undergo an active, energy-dependent process leading to a cascade of biochemical and morphologic events that result in the irreversible fragmentation of genomic DNA and then of the cell itself. Biochemical and morphological studies have demonstrated that the involution of the normal, hyperplastic and cancerous prostate after castration is the result of programmed cell death. Numerous group have studied the role of calcium in programmed cell death's triggering. In androgen-independent prostatic cell it had been proved that it was possible to trigger programmed cell death by inducing a sustained elevation of the intracellular calcium. Actually, some features tend to explain more precisely the exact role of calcium in apoptosis of prostatic cells.

Apoptosis↗
PubMed1993

Influence of Ca-activated brevin on the mechanical properties of skinned smooth muscle.

Solutions of purified brevin were applied to skinned thin bundles or isolated fibres of smooth muscle. This produced a sharp drop of isometric tension, an effect due to the severing effect of brevin on actin filaments, partially depleted from tropomyosin in skinned preparations. On skinned single fibres, brevin accelerates the speed of unloaded shortening. As no effect was detected on the myofibrillar ATPase turnover rate, brevin was thought to affect the viscosity of the cytoplasm. This was confirmed by analysis of the cytoplasm stiffness which decreased in the presence of brevin. It is proposed that Ca-activated brevin acts on actin-filamin gels, set in parallel to the contractile apparatus.

Adenosine Triphosphatases↗
PubMed1993

Lack of dystrophin but normal calcium homeostasis in smooth muscle from dystrophic mdx mice.

The free cytosolic Ca2+ concentration ([Ca2+]i) in the dystrophin-lacking smooth muscle from mdx mice was studied to gain new insights into the relation between dystrophin and cytoplasmic Ca2+ homeostasis, which was reported to be impaired in the mdx skeletal muscle. We observed that [Ca2+]i, as measured with the fluorescent Ca2+ indicator fura-2, was not elevated in resting smooth muscle of the vas deferens from mdx mice, in comparison with control C57 mice. Changes of the external Ca2+ concentration evoked similar changes of [Ca2+]i in mdx and control vas deferens. During contraction, cytosolic Ca2+ transients were identical, both in amplitude and in kinetics, whether or not dystrophin was present. Stretches evoked similar Ca2+ increases in muscles from both strains. Intracellular Ca2+ homeostasis appears to be unimpaired in mdx smooth muscle. Thus, the lack of dystrophin per se does not automatically induce a perturbation of Ca metabolism in muscle cells.

Animals↗
PubMed1993

Critical evaluation of cytosolic calcium determination in resting muscle fibres from normal and dystrophic (mdx) mice.

The fluorescent probe Fura-2/AM was used to determine cytosolic free calcium concentration in soleus muscle and in isolated flexor digitorum brevis fibres. This required a precise calibration; therefore, each calibration parameter was studied in situ. The influence of the dye concentration on calcium measurements was also examined. This precise calibration technique was used to compare absolute free calcium concentration in resting preparations from dystrophic (mdx) and control (C57) mice. We showed that the behavior of the dye was not similar in C57 and in mdx muscles. For this reason, we did not confirm the previous results that cytosolic free calcium concentration is increased in mdx muscles.

Animals↗
PubMed1993

ATP induced-relaxation in the mouse bladder smooth muscle.

1. The effect of adenosine 5'-triphosphate (ATP) on the free cytosolic Ca2+ concentration ([Ca2+]i) as measured with the fluorescent Ca(2+)-indicator fura-2, and on force was investigated in the intact smooth muscle strips of the mouse urinary bladder. 2. ATP elicited, when exogenously applied, a large increase of [Ca2+]i with limited force development resulting in a marked Ca(2+)-force dissociation. 3. Release of endogenous neurotransmitters by transmural electrical stimulation (TES) for 30 s induced a steady increase of [Ca2+]i and a peak contraction, followed within 15 s by a relaxation. 4. In carbachol-prestimulated preparations, ATP elicited an initial rise of [Ca2+]i followed by a return to the initial precontraction Ca(2+)-level. Force in contrast presented a biphasic pattern, i.e. an initial contraction was followed by a sustained relaxation. 5. In the K(+)-depolarized precontracted preparation, ATP elicited a slight initial rise of [Ca2+]i. The partial relaxation of the force during depolarization was not preceded by a transient contraction. 6. The ATP-induced relaxation of the K(+)-prestimulated preparations was not inhibited by 8-phenyltheophylline, a potent P1-purinoceptor antagonist. 7. The order of potency for relaxation of the ATP analogues was 2-MeSATP > ATP > beta gamma Me-ATP, which is characteristic for P2y-purinoceptors. 8. These results indicate that, besides its activating effect, ATP also relaxes the mouse urinary bladder. It is suggested that the relaxant effect, mediated through P2y-purinoceptors, is mainly responsible for the low contractile potency of ATP in the bladder.

Adenosine Triphosphate↗
PubMed1993

Post-receptor pathway of the ATP-induced relaxation in smooth muscle of the mouse vas deferens.

1. The post-receptor pathway of the ATP relaxant effect in K(+)-precontracted vas deferens smooth muscle (VD) was examined. 2. The relaxation to ATP was not antagonized either by 10 microM methylene blue, a cyclic GMP inhibitor, by 10 microM indomethacin, an inhibitor of prostaglandin synthesis or by 100 microM NG-nitro-L-arginine, an inhibitor of NO production. 3. The Rp-diastereomer of adenosine 3':5'-cyclic monophosphorothioate (Rp-cAMPS) 200 microM, a competitive inhibitor of cyclic AMP significantly diminished the relaxant response to ATP. 4. Isoprenaline 10 microM, a beta-adrenoceptor agonist, produced a sustained relaxation, inhibited by Rp-cAMPS, without a significant change in [Ca2+]i, thereby mimicking the ATP-induced relaxant effect. 5. The level of the phosphorylated myosin light chain in the precontracted VD was significantly lowered by 1000 microM ATP. 6. ATP (1000 microM) and isoprenaline (10 microM) produced the same increase (+ 50%) of [cyclic AMP] when applied to a resting VD. 7. The effect of simultaneous increases of [Ca2+]i and of [cyclic AMP] produced by externally applied ATP are discussed. 8. These results suggest that ATP-induced relaxation in K(+)-precontracted VD is mediated by the activation of adenylyl cyclase.

Adenosine Triphosphate↗