Biomedical subjects

B Allard

Publications and source records attributed to B Allard.

At least 19 recordsLinked to original sources

PubMed2006

In vitro antiretroviral activity and in vitro toxicity profile of SPD754, a new deoxycytidine nucleoside reverse transcriptase inhibitor for treatment of human immunodeficiency virus infection.

SPD754 (AVX754) is a deoxycytidine analogue nucleotide reverse transcriptase inhibitor (NRTI) in clinical development. These studies characterized the in vitro activity of SPD754 against NRTI-resistant human immunodeficiency virus type 1 (HIV-1) and non-clade B HIV-1 isolates, its activity in combination with other antiretrovirals, and its potential myelotoxicity and mitochondrial toxicity. SPD754 was tested against 50 clinical HIV-1 isolates (5 wild-type isolates and 45 NRTI-resistant isolates) in MT-4 cells using the Antivirogram assay. SPD754 susceptibility was reduced 1.2- to 2.2-fold against isolates resistant to zidovudine (M41L, T215Y/F, plus a median of three additional nucleoside analogue mutations [NAMs]) and/or lamivudine (M184V) and was reduced 1.3- to 2.8-fold against isolates resistant to abacavir (L74V, Y115F, and M184V plus one other NAM) or stavudine (V75T/M, M41L, T215F/Y, and four other NAMs). Insertions at amino acid position 69 and Q151M mutations (with or without M184V) reduced SPD754 susceptibility 5.2-fold and 14- to 16-fold, respectively (these changes gave values comparable to or less than the corresponding values for zidovudine, lamivudine, abacavir, and didanosine). SPD754 showed similar activity against isolates of group M HIV-1 clades, including A/G, B, C, D, A(E), D/F, F, and H. SPD754 showed additive effects in combination with other NRTIs, tenofovir, nevirapine, or saquinavir. SPD754 had no significant effects on cell viability or mitochondrial DNA in HepG2 or MT-4 cells during 28-day exposure at concentrations up to 200 microM. SPD754 showed a low potential for myelotoxicity against human bone marrow. In vitro, SPD754 retained activity against most NRTI-resistant HIV-1 clinical isolates and showed a low propensity to cause myelotoxicity and mitochondrial toxicity.

Anti-HIV Agents↗

PubMed2004

Differential effects of deoxycholic acid and taurodeoxycholic acid on NF-kappa B signal transduction and IL-8 gene expression in colonic epithelial cells.

Several effects of bile acids (BAs) on colonic epithelial cells (CECs) have been described, including induction of proliferation and apoptosis. Some of these effects are mediated through activation of the NF-kappa B transcriptional system. In this study, we investigated the molecular mechanisms underlying the BA-induced gene expression in CECs. The human CEC line HT-29 and primary human CECs were treated with dilutions of salts of deoxycholic acid (DCA) and taurodeoxycholic acid (TDCA). NF-kappa B binding activity was analyzed with EMSA, RelA translocation with immunofluorescence, and I kappa B alpha- and RelA-phosphorylation with Western blot analysis. IL-8 mRNA and protein expression were assessed by quantitative PCR and ELISA. Functional impact of NF-kappa B activation was determined by blocking the proteasome activity with MG132 or by preventing IKK activity with a dominant-negative IKK beta delivered by adenoviral dominant-negative (dn) IKK beta (Ad5dnIKK beta). DCA and TDCA induced IL-8 expression in a dose- and time-dependent manner. It is interesting that DCA but not TDCA induced I kappa B alpha-phosphorylation, RelA translocation, and NF-kappa B binding activity. Accordingly, the proteasome inhibitor MG132 blocked DCA- but not TDCA-induced IL-8 gene expression. In contrast, TDCA-induced IL-8 gene expression correlated with enhanced RelA phosphorylation, which was blocked by Ad5dnIKK beta. Our data suggest that DCA-induced signal transduction mainly utilized the I kappa B degradation and RelA nuclear translocation pathway, whereas TDCA primarily induced IL-8 gene expression through RelA phosphorylation. These differences may have implications for the understanding of the pathophysiology of inflammation and carcinogenesis in the gut.

Bile Acids and Salts↗

PubMed2004

NF-kappaB inducing kinase activates NF-kappaB transcriptional activity independently of IkappaB kinase gamma through a p38 MAPK-dependent RelA phosphorylation pathway.

Molecular and biochemical analysis indicates that nuclear transcription factor kappaB (NF-kappaB)-inducing kinase (NIK) mediates IKK activation and NF-kappaB transcriptional activity. However, gene deletion studies suggest that NIK triggers gene expression without affecting IkappaBalpha degradation and NF-kappaB DNA binding activity. In order to investigate the role of NIK in NF-kappaB transcriptional activity, we used mouse embryonic fibroblasts (MEF) derived from wild-type (wt) and IkappaB kinase gamma (IKKgamma) gene deficient (IKKgamma(-/-)) mice. We report that although TNF-induced NF-kappaB transcriptional activity is abolished in IKKgamma(-/-) cells, adenoviral gene delivery of NIK (Ad5NIK) still enhanced transcriptional activity and IL-6 mRNA accumulation. Moreover, NIK targets the transactivation function of NF-kappaB through stimulation of the transactivation domain (TAD) of RelA (S536) in IKKgamma(-/-) cells. Interestingly, Ad5NIK, but not TNF, induces RelA S536 and p38 mitogen-activated protein kinase (MAPK) phosphorylation in IKKgamma(-/-) cells. Functional analysis demonstrated that Ad5NIK-induced NF-kappaB transcriptional activity, IL-6 mRNA expression and RelA phosphorylation are inhibited by the p38 inhibitor SB203580, suggesting a role for this MAPK in NIK signaling to NF-kappaB. These data demonstrate for the first time the presence of an IKKgamma-independent NIK/p38 MAPK-dependent signaling pathway that activates NF-kappaB and induces pro-inflammatory gene expression through RelA phosphorylation.

Animals↗

PubMed2001

High molecular weight lipids from the trilaminar outer wall (TLS)-containing microalgae Chlorella emersonii, Scenedesmus conmmunis and Tetraedron minimum.

High molecular weight lipids were isolated from Chlorella emersonii, Scenedesmus communis and Tetraedron minimum, thin trilaminar outer wall (TLS)-containing freshwater microalgae producing an insoluble non-hydrolysable biopolymer (i.e. algaenan). Molecular weight determination by gel permeation chromatography indicated that their molecular weights range from ca. 400 to 2000 Da. Flash pyrolysis with in situ methylation using tetramethylammonium hydroxide (TMAH) and alkaline hydrolysis showed that the high molecular weight lipids isolated from C. emersonii and S. communis are mainly composed of saturated n-C26 and n-C28 fatty acids and alcohols and of saturated n-C30 and n-C32 alpha,omega-diols and omega-hydroxy acids. In contrast the high molecular weight lipids isolated from T. minimum are predominantly composed of long-chain fatty acids and omega-hydroxy acids. Aromatic moieties were also identified in small amounts in the thermochemolysate and in the hydrolysate. Chemical structural models containing long-chain mono- and polyesters were proposed for the high molecular weight lipids isolated from the three microalgae in agreement with analytical and spectroscopic data. Structural similarity between the outer cell wall of these microalgae and the cuticular membrane of higher plants is suggested.

Cell Wall↗

PubMed2000

Characterization and gene expression of high conductance calcium-activated potassium channels displaying mechanosensitivity in human odontoblasts.

Odontoblasts form a layer of cells responsible for the dentin formation and possibly mediate early stages of sensory processing in teeth. Several classes of ion channels have previously been identified in the odontoblast or pulp cell membrane, and it is suspected that these channels assist in these events. This study was carried out to characterize the K(Ca) channels on odontoblasts fully differentiated in vitro using the patch clamp technique and to investigate the HSLO gene expression encoding the alpha-subunit of these channels on odontoblasts in vivo. In inside-out patches, K(Ca) channels were identified on the basis of their K(+) selectivity, conductance, voltage, and Ca(2+) dependence. In cell-attached patches, these channels were found to be activated by application of a negative pressure as well as an osmotic shock. By reverse transcription-polymerase chain reaction, a probe complementary to K(Ca) alpha-subunit mRNA was constructed and used for in situ hybridization on human dental pulp samples. Transcripts were expressed in the odontoblast layer. The use of antibodies showed that the K(Ca) channels were preferentially detected at the apical pole of the odontoblasts. These channels could be involved in mineralization processes. Their mechanosensitivity suggests that the fluid displacement within dentinal tubules could be transduced into electrical cell signals.

Adolescent↗

PubMed2000

Elevated subsarcolemmal Ca2+ in mdx mouse skeletal muscle fibers detected with Ca2+-activated K+ channels.

Duchenne muscular dystrophy results from the lack of dystrophin, a cytoskeletal protein associated with the inner surface membrane, in skeletal muscle. The cellular mechanisms responsible for the progressive skeletal muscle degeneration that characterizes the disease are still debated. One hypothesis suggests that the resting sarcolemmal permeability for Ca(2+) is increased in dystrophic muscle, leading to Ca(2+) accumulation in the cytosol and eventually to protein degradation. However, more recently, this hypothesis was challenged seriously by several groups that did not find any significant increase in the global intracellular Ca(2+) in muscle from mdx mice, an animal model of the human disease. In the present study, using plasma membrane Ca(2+)-activated K(+) channels as subsarcolemmal Ca(2+) probe, we tested the possibility of a Ca(2+) accumulation at the restricted subsarcolemmal level in mdx skeletal muscle fibers. Using the cell-attached configuration of the patch-clamp technique, we demonstrated that the voltage threshold for activation of high conductance Ca(2+)-activated K(+) channels is significantly lower in mdx than in control muscle, suggesting a higher subsarcolemmal [Ca(2+)]. In inside-out patches, we showed that this shift in the voltage threshold for high conductance Ca(2+)-activated K(+) channel activation could correspond to a approximately 3-fold increase in the subsarcolemmal Ca(2+) concentration in mdx muscle. These data favor the hypothesis according to which an increased calcium entry is associated with the absence of dystrophin in mdx skeletal muscle, leading to Ca(2+) overload at the subsarcolemmal level.

Animals↗

PubMed2000

Comparison of neutral lipid profile of various trilaminar outer cell wall (TLS)-containing microalgae with emphasis on algaenan occurrence.

The neutral lipid profiles of nine species of thin trilaminar outer wall (TLS)-containing freshwater and marine microalgae from the class of Chlorophyceae were studied with emphasis on the relationship between the lipid content and the occurrence of insoluble non-hydrolysable biopolymer (i.e. algaenan). All the freshwater microalgae produce a highly aliphatic algaenan. In sharp contrast, no algaenan was isolated from the two marine microalgae, Chlorella marina and Chlorella minutissima marina, supporting the absence of a close relationship between the presence of TLS and the occurrence of algaenan. High molecular weight straight-chain hydrocarbons (C23-C29) were identified in most of the algaenan-producing microalgae and in the algaenan-devoid C. minutissima marina, whereas only low molecular weight hydrocarbons were detected in algaenan-producing Scenedesmus subspicatus and in algaenan-devoid C. marina. Sterols, phytol and fatty alcohols were the major constituents of the polar fraction of the neutral lipids of all the microalgae investigated. High molecular weight saturated or mono-unsaturated alcohols were detected in C. emersonii and in all the microalgae belonging to the genus Scenedesmus. High amounts of saturated C30 and C32 alpha,omega-diols were also detected in S. subspicatus, S. armatus and S. pannonicus. Three classes of lipids were encountered in very small amounts in the medium polarity fraction of the neutral lipids of the microalgae investigated: (i) Monoesters composed predominantly of saturated C16 or C18 fatty acids and saturated C8, C16 or C18 alcohols and (ii) long-chain methyl ketones from C25 to C31 were detected in several species and (iii) methyl esters of fatty acids ranging from C16 to C28 were identified in all the microalgae. Attempts to use the neutral lipid composition and particularly the unusual long-chain lipids, as specific indicators of the occurrence of algaenan in TLS-containing microalgae were unsuccessful.

Alcohols↗

PubMed2000

Stretch-induced activation of Ca(2+)-activated K(+) channels in mouse skeletal muscle fibers.

High-conductance Ca(2+)-activated K(+) (K(Ca)) channels were studied in mouse skeletal muscle fibers using the patch-clamp technique. In inside-out patches, application of negative pressure to the patch induced a dose-dependent and reversible activation of K(Ca) channels. Stretch-induced increase in channel activity was found to be of the same magnitude in the presence and in the absence of Ca(2+) in the pipette. The dose-response relationships between K(Ca) channel activity and intracellular Ca(2+) and between K(Ca) channel activity and membrane potential revealed that voltage and Ca(2+) sensitivity were not altered by membrane stretch. In cell-attached patches, in the presence of high external Ca(2+) concentration, stretch-induced activation was also observed. We conclude that membrane stretch is a potential mode of regulation of skeletal muscle K(Ca) channel activity and could be involved in the regulation of muscle excitability during contraction-relaxation cycles.

Adenosine Triphosphate↗

PubMed1999

Intracellular calcium signals measured with indo-1 in isolated skeletal muscle fibres from control and mdx mice.

1. Intracellular free calcium concentration ([Ca2+]i) was measured with the fluorescent indicator indo-1 in single skeletal fibres enzymatically isolated from the flexor digitorum brevis and interosseus muscles of control and dystrophic mdx C57BL/10 mice. Measurements were taken from a portion of fibre that was voltage clamped to allow detection of depolarization-induced changes in [Ca2+]i. 2. The mean (+/- s.e.m.) initial resting [Ca2+]i from all control and mdx fibres tested was 56 +/- 5 nM (n = 72) and 48 +/- 7 nM (n = 57), respectively, indicating no significant overall difference between the two groups. However, when comparing a batch of control and mdx fibres obtained from mice older than approximately 35 weeks, resting [Ca2+]i was significantly lower in mdx (16 +/- 4 nM, n = 11) than in control fibres (71 +/- 10 nM, n = 14). 3. Changes in [Ca2+]i elicited by short (5-35 ms) depolarizing pulses from -80 to 0 mV showed similar properties in control and mdx fibres. After a 5 ms duration pulse the mean time constant of [Ca2+]i decay was, however, significantly elevated in mdx as compared to control fibres, by a factor of 1.5-2. For longer pulses, no significant difference could be detected. 4. In response to 50 ms duration depolarizing pulses of various amplitudes the threshold for detection of an [Ca2+]i change and the peak [Ca2+]i reached for a given potential were similar in control and mdx fibres. 5. Overall results show that mdx skeletal muscle fibres are quite capable of handling [Ca2+]i at rest and in response to membrane depolarizations.

Age Factors↗

PubMed1999

Determination of the molecular weight of fulvic acids by UV/VIS spectroscopy

Partial least squares (PLS) modeling was applied to investigate number-average molecular weights (Mn) and weight-average molecular weights (Mw) of fulvic acids (FAs) in relation to the corresponding UV/VIS spectra. The Mn and Mw values were determined by size exclusion chromatography (SEC). The impact of pH control, wavelength range and density as well as smoothing and derivation of spectra were tested. It was found that PLS models based on absorbance spectra can be a fast and powerful complement to existing techniques employed for determination of molecular weights of FAs. Control of pH of the FA solutions is important for the performance of the models. The models were also compared with the best univariate alternatives.

Journal Article↗

PubMed1999

Dependence of superoxide anion production on extracellular and intracellular calcium ions and protein kinase C in PMA-stimulated bovine neutrophils.

The involvement of both intracellular and extracellular calcium, as well as the activation of protein kinase C (PKC), in phorbol myristate acetate (PMA)-stimulated respiratory burst in bovine neutrophils has been studied. PMA significantly stimulated the superoxide anion production by these cells. The increased production of superoxide anion was inhibited by BAPTA/AM, an intracellular calcium ([Ca2+]i) chelator, but not affected by EGTA, an extracellular calcium ([Ca2+]0) chelator. PMA also induced PKC activation, and a PKC inhibitor, calphostin C, blocked the stimulatory effect of PMA on superoxide anion production by the neutrophils. Therefore, we conclude that PMA-induced respiratory burst in bovine neutrophils is [Ca2+]i- but not [Ca2+]0-dependent, and also requires PKC activation.

Animals↗

PubMed1998

Activation of Ca2+-activated K+ channels by an increase in intracellular Ca2+ induced by depolarization of mouse skeletal muscle fibres.

1. Ionic currents were simultaneously recorded at macroscopic and unitary level using the whole-cell and cell-attached patch-clamp procedures together on the same portion of isolated mouse skeletal muscle fibres. 2. In the presence of Tyrode solution in the patch pipette and Tyrode-TTX solution in the bath, macroscopic and unitary currents through delayed rectifier K+ channels were simultaneously recorded in response to depolarizing pulses of 1 s duration. 3. In five fibres, successive long-lasting incremental depolarizing levels induced, at -40 mV or -30 mV, the opening of a high conductance channel carrying an outward current superimposed on delayed rectifier K+ channel activity. Opening of this high conductance channel was not observed when the depolarization steps were applied in the patch pipette. 4. Using the same depolarizing protocol, activation of a high conductance channel was also observed in two fibres in the presence of a K+-rich solution in the pipette (145 mM K+) . 5. With either Tyrode or K+-rich solution in the pipette, unitary current amplitudes of the high conductance channel matched well with the values obtained for Ca2+-activated K+ (KCa) channels in inside-out patches under similar ionic conditions. 6. Indo-1 fluorescence measurements showed that the stimulation protocol that led to KCa channel opening induced stepwise increases in intracellular [Ca2+] in the submicromolar range. 7. Our results provide evidence that activation of sarcolemmal KCa channels can be induced by a rise in intracellular [Ca2+] following voltage-activated sarcoplasmic reticulum Ca2+ release.

Animals↗

PubMed1997

Similarity of ATP-dependent K+ channels in skeletal muscle fibres from normal and mutant mdx mice.

1. ATP-dependent K+ (KATP) channels were studied in fibres isolated from flexor digitorum brevis and interosseal skeletal muscles of normal and mutant mdx mice using the patch clamp technique in the presence of asymmetrical K+ concentrations (5 mM K+ in the pipette and in vivo intracellular [K+] or 145 mM K+ at the cytoplasmic face). 2. In cell-attached patches from mdx muscle fibres bathed in K(+)-rich solution, cell poisoning with fluorodinitrobenzene induced partially reversible opening of channels carrying an outward current of an amplitude of 1.2 pA at 0 mV. Exposure of fibres to the K+ channel opener cromakalim led to opening of the same type of channel. These channels were assumed to be KATP channels. 3. On excision of inside-out patches from mdx muscle fibres, in the absence of intracellular ATP, KATP channels were active: they carried a unitary outward current of 1.6 pA at 0 mV and were inhibited by intracellular ATP and glibenclamide. The number of KATP channels per patch was not significantly different in muscles from normal and mdx mice. 4. In inside-out patches, in the presence of 1 mM intracellular Mg2+, slope conductances of 21 and 20.3 pS were found for KATP channels in normal and mdx muscle, respectively. In the absence of Mg2+, slope conductances of KATP channels were 31.3 and 32 pS in normal and mdx muscle, respectively and KATP channel activity was augmented in mdx muscle in the same way as in normal muscle. Activity of the same KATP channel was observed in extensor digitorum longus muscle from normal and mdx mice. 5. In inside-out patches held at 0 mV, the relationship between KATP channel activity and intracellular ATP was described by a Hill equation: Ki values were 23 and 21 microM and Hill coefficients were 1.8 and 1.9 in normal and mdx muscle, respectively. 6. These results indicate that the distribution, the conductance properties and ATP sensitivity of KATP channels do not differ in normal and in mdx mouse skeletal muscle.

Adenosine Triphosphate↗

PubMed1996

Intracellular Ca2+ changes and Ca2+-activated K+ channel activation induced by acetylcholine at the endplate of mouse skeletal muscle fibres.

1. Enzymatically isolated skeletal muscle fibres were used to investigate the effects of applying acetylcholine (ACh) onto the endplate area on intracellular free calcium concentration ([Ca2+]i) measured using the indicator indo-1 and single channel activity using the patch clamp technique. 2. Using a Tyrode solution containing 5 microM tetrodotoxin (TTX) as extracellular solution, ACh applications (at 0.1 or 1 mM) onto the endplate induced intracellular free calcium transients the mean maximal amplitude of which was 360 +/- 30 nM from a mean resting value of 72 +/- 7 nM (n = 13). In cells bathed with a K(+)-rich solution (145 mM K+), applications of ACh (0.1 mM) induced transient rises in [Ca2+]i from a mean resting value of 53 +/- 7 nM to a maximum of 222 +/- 24 nM (n = 33). 3. In cell-attached membrane patches at the endplate membrane of muscle fibres bathed in a K(+)-rich external solution, using a pipette filled with Tyrode solution, external application of 0.1 mM ACh could induce a transient burst opening of channels carrying an outward current of an average amplitude of 4.6 +/- 0.2 pA at 0 mV (n = 8). 4. These channels were characterized as Ca2(+)-activated K+ channels. At 0 mV, in inside-out patches excised from the endplate membrane area, they displayed a conductance of 60 and 224 pS in the presence of Tyrode and K(+)-rich solution in the pipette, respectively. Half-maximum activation was found for a [Ca2+]i close to 4 microM. The channels showed a typical voltage dependence. In outside-out patches these channels were shown to be blocked by 100 nM charybdotoxin (CTX). 5. In fibres bathed in a Tyrode solution containing TTX (5 microM), CTX had no clear effect on the change in membrane voltage, recorded near the endplate with a single intracellular microelectrode, in response to the application of ACh. 6. Although the physiological relevance of this ACh-induced K+ channel activation remains unclear, results suggest that, in the presence of a physiological extracellular [Ca2+], Ca2+ entry through the endplate nicotinic receptors can produce a local increase in [Ca2+]i, sufficient to trigger the opening of Ca2+-activated K+ channels in the adjacent surface membrane.

Acetylcholine↗

PubMed1995

Dose-dependent activation and block by bisG10, a K+ channel blocker, of mouse and frog skeletal muscle KATP channels.

The effects of a K+ channel blocker, bisG10, were examined on ATP-sensitive K+ (KATP) channels in membrane patches excised from mammalian and amphibian skeletal muscle fibres using the patch-clamp technique. At micromolar concentrations, bisG10, added on the intracellular side, induced a strong, reversible, flickery block of KATP channels. BisG10, added on the extracellular side, was about 100-fold less potent at inhibiting channel activity. At 10 nM, intracellular bisG10 increased KATP channel activity. This activation was independent of the presence of internal ATP or Mg2+. The inhibitory effect of bisG10 most likely arose from open-channel block whereas activation could result from more complex, indirect interactions.

Adenosine Triphosphate↗

PubMed1995

Protonophoric activity of ellipticine and isomers across the energy-transducing membrane of mitochondria.

Ellipticine is an antitumor alkaloid capable of uncoupling mitochondrial oxidative phosphorylation. It behaves as a lipophilic weak base with pK = 7.40. We have investigated its molecular mode of action using several of its isomers with pK ranging between 5.8 and 7.7 and ellipticinium, which is a permanent cationic derivative. The effects of these molecules on mitochondrial oxygen uptake and transmembrane potential were compared at different pHs. Ellipticinium exhibited very low effects on both respiratory rate and membrane potential. By contrast, protonable derivatives showed maximal stimulation of oxygen uptake and depolarizing effects when the pH of the medium was close to the drug pK. These effects were lowered when the transmembrane delta pH was dissipated, which indicates that the neutral form of the drug is implicated in the uncoupling mechanism. In addition, protonable derivatives of ellipticine display a linear relationship between oxidation rate and transmembrane potential, which suggests that the uncoupling properties of these molecules result from a protonophoric mechanism. From these results, the following cyclic protonophoric mechanism is proposed for protonable ellipticines: (i) electrophoretical accumulation of the protonated form; (ii) deprotonation at the matrix interface; (iii) diffusion outwards; and (iv) reprotonation at the external interface.

Animals↗

PubMed1995

Activation of ATP-dependent K+ channels by metabolic poisoning in adult mouse skeletal muscle: role of intracellular Mg(2+) and pH.

1. The effects of metabolic poisoning, intracellular Mg(2+) and pH on ATP-dependent K+ (K+ATP) channels were examined in adult mouse isolated skeletal muscle fibres using the patch clamp technique. 2. In cell-attached membrane patches, while openings of one kind of channel could only rarely be detected under control conditions, cell poisoning with fluorodinitrobenzene (FDNB), dinitrophenol (DNP) and cyanide (CN) induced a strong and partially reversible increase in channel activity. 3. Slope conductance and glibenclamide sensitivity of this outward current indicated that the channel activated during poisoning was the K+ATP channel. 4. Single channel current amplitude was reduced during poisoning, but remained unchanged when activation of the K+ATP channel was induced by cromakalim. 5. In inside-out membrane patches, in the absence of intracellular ATP, intracellular application of Mg2+ decreased channel activity and single channel current amplitude. Inhibition of K+ATP channels by ATP was also reduced. 6. In the absence of intracellular ATP, a decrease in intracellular pH induced a reduction in channel activity and single channel current amplitude. Inhibition of K+ATP channels by ATP was also reduced. 7. The reduction of single channel current amplitude during poisoning was attributed to an increase in intracellular Mg2+ concentration caused by a fall in intracellular ATP concentration. These results also show that metabolic poisoning causes direct activation of K+ATP channels in skeletal muscle, and that is activation is at least partially mediated through an increase in intracellular Mg(2+) concentration and a decrease in intracellular pH.

2,4-Dinitrophenol↗

PubMed1995

Coexistence of two classes of glibenclamide-inhibitable ATP-regulated K+ channels in avian skeletal muscle.

Avian skeletal muscle expresses two types of ATP-sensitive K+ channels which have a unitary conductance of 15pS. These K+ channels can be distinguished pharmacologically by their high or low sensitivity to the antidiabetic sulphonylurea blocker glibenclamide. Both channels are activated by the K+ channel opener cromakalim. Chick skeletal muscle expresses high-affinity binding sites for [3H]glibenclamide (Kd = 0.6nM) which presumably correspond to the ATP-sensitive K+ channels with the greatest sensitivity to glibenclamide. The density of these high-affinity binding sites varies during muscle development. The maximum density (500fmol/mg protein) appears at 16 days in ovo, i.e. at a period when myoblasts have differentiated into myotubes and when innervation of myotubes has started. After this maximum, the level of [3H]glibenclamide-binding sites decreases to a plateau value of 100fmol/mg protein at 2-5 days post-natal. When muscle cells are put in cultures, the high-affinity binding sites disappear rapidly. Neither glibenclamide nor cromakalim have any effect on normal physiological chick muscle contraction. They have no effect on contracture and/or 86Rb+ efflux produced by metabolic poisoning.

Adenosine Triphosphate↗