Immunolocalization of annexins IV, V and VI in the failing and non-failing human heart.
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The failing human heart is characterized by changes in the expression and function of proteins involved in intracellular Ca2+ cycling, resulting in altered Ca2+ transients and impaired contractile properties of cardiac muscle. The role of the cardiac annexins in this process remains unclear. Annexins may play a role in the regulation of Ca2+ pumps and exchangers on the sarcolemma, and have been shown to be altered in some cardiac disease states. OBJECTIVE: The goal of this study was to compare the immunolocalization and expression of annexins IV, V and VI in failing and non-failing human hearts. METHODS: We used immunostaining to identify the subcellular location of annexins IV, V and VI proteins within the myocardial cell, and Western blot analysis to quantify the proteins in the same hearts. RESULTS: Annexin IV showed a cytoplasmic distribution in both failing and non-failing human heart cells. Annexin V was localized at the z-line, around lipofuscin granules, and in the cytosol in the non-failing heart cells. Annexin VI was localized at the sarcolemma and intercalated disc. Protein levels of annexins IV and V were up-regulated in failing human hearts, while the expression of annexin VI was unchanged. CONCLUSIONS: Alterations in the intracellular localization of annexins, along with up-regulation of annexins IV and V in the failing human heart cells, suggests differential regulation of these Ca2+ regulatory proteins during heart failure. (+info)
Immunochemical evidence for a unique GPI-anchored carbonic anhydrase isozyme in human cardiomyocytes.
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To clarify the controversial question of cell-specific distribution of carbonic anhydrase (CA) in the heart, endothelial cells and cardiomyocytes were isolated from porcine and human hearts and were characterized with cell-specific markers. CA activity was found in the microsomal fraction of both cell types. It was shown by Triton X-114 phase separation that both cell types possess a membrane-bound form of CA. These CAs share the same mechanism of membrane-anchoring via glycosylphosphatidylinositol (GPI), which excludes identity with transmembrane isoforms CA IX or CA XII. Western blotting analysis of human microsomes with anti-human CA IV antibodies revealed a marked difference in immunoreactivity. Endothelial CA activity resulted in 11-fold stronger CA IV bands compared with identical amounts of myocytic CA activity, indicating that cardiac endothelium and cardiomyocytes possess immunologically distinct forms of CA. We conclude that in human hearts CA IV is associated with the endothelium, whereas most of the CA in myocytes is not identical with one of the known CA isozymes. This suggests that cardiomyocytic CA is a novel isozyme. (+info)
Generation of intracellular pH gradients in single cardiac myocytes with a microperfusion system.
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This study describes the use of a microperfusion system to create rapid, large regional changes in intracellular pH (pH(i)) within single ventricular myocytes. The spatial distribution of pH(i) in single myocytes was measured with seminaphthorhodafluor-1 fluorescence using confocal imaging. Changes in pH(i) were induced by local external application of NH(4)Cl, CO(2), or sodium propionate. Local application was achieved by simultaneously directing two parallel square microstreams, each 275 microm wide, over a single myocyte oriented perpendicular to the direction of flow. One stream contained the control solution, and the other contained a weak acid or base. End-to-end, stable pH(i) gradients as large as 1 pH unit were readily created with this technique. This result indicates that pH within a single cardiac cell may not always be spatially uniform, particularly when weak acid or base gradients are present, which can occur, for example, in regional myocardial ischemia. The microperfusion method should be useful for studying the effects of localized acidosis on myocyte function, estimating intracellular ion diffusion rates, and, possibly, inducing regional changes in other important intracellular ions. (+info)
Selective enhancement of beta-adrenergic receptor signaling by overexpression of adenylyl cyclase type 6: colocalization of receptor and adenylyl cyclase in caveolae of cardiac myocytes.
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We investigated the effect of adenovirally mediated overexpression of adenylyl cyclase type 6 (AC6), a major form of AC expressed in mammalian heart, on G protein-coupled receptor regulation of cAMP production in neonatal rat ventricular myocytes. Following gene transfer of AC6, isoproterenol- and forskolin-stimulated increases in cAMP were markedly enhanced, whereas basal levels of cAMP and responses to several other agonists that stimulate cAMP formation, e. g., prostaglandin E(2) (PGE(2)), H(2) agonist, glucagon, and A(2) agonist were not increased. Studies to test whether the selective enhancement in beta-adrenergic receptor (AR) response might result from inhibition of AC6 by Galpha(i) and Gbetagamma indicated that pertussis toxin-sensitive inhibition by the muscarinic cholinergic agonist carbachol was unaltered in myocytes overexpressing AC6. Pertussis toxin treatment failed to reveal an enhancement by AC6 overexpression of basal or PGE(2)-stimulated cAMP. Immunoblot analysis of membrane fractions indicated that beta(1)-AR and AC6 are expressed in fractions enriched in caveolin-3 and morphologic caveolae. The data suggest that loss of G(i)-mediated inhibition is not the mechanism for enhancement of beta-AR-stimulated cAMP formation and that key components of beta-AR-mediated activation of AC exist in caveolae of cardiac myocytes, providing a means by which beta-AR response is selectively enhanced by increasing AC6 expression. (+info)
Elevated subsarcolemmal Ca2+ in mdx mouse skeletal muscle fibers detected with Ca2+-activated K+ channels.
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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. (+info)
Acute ethanol treatment decreases intracellular calcium-ion transients in mouse single skeletal muscle fibres in vitro.
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Alcohol misuse frequently leads to muscle weakness, which may also occur in the setting of acute and chronic alcoholic myopathies. At the cellular level, ethanol has been found to interfere with signalling mechanisms in cardiac myocytes, skeletal myotubes, and smooth muscle cells. In this study, we focused on the effects of ethanol on the intracellular calcium ([Ca(2+)](i)) transients responsible for excitation-contraction (EC) coupling in isolated mouse skeletal fibres loaded with the fluorescent Ca(2+) indicator fura-2. Following electrical stimulation, ethanol caused a significant reversible dose-dependent reduction in [Ca(2+)](i) transient amplitude, already significant at 100 mM ethanol (P = 0.03), without modifying resting [Ca(2+)](i). Evaluating the potential loci for the effects of ethanol, we indirectly measured sarcolemmal Ca(2+) entry by monitoring Mn(2+)-quenching of intracellular fura-2 via the nitrendipine-sensitive Ca(2+) channels during electrical pacing. Ethanol at doses of 20 mM and greater caused a dose-dependent reduction in the rate of fura-2 quenching (all at P<0.05). Moreover, the intracellular pool of Ca(2+) releasable by caffeine was found to be reduced at a minimum of 300 mM ethanol (P = 0.05). We conclude that ethanol reduces the [Ca(2+)](i) transients underlying EC coupling in single mouse skeletal muscle fibres. This acute effect of ethanol was primarily due to an inhibitory effect of ethanol on sarcolemmal Ca(2+) influx via voltage-operated Ca(2+)-channels and, to a lesser extent, to a reduction in the Ca(2+) sarcoplasmic reticulum loading state. This inhibitory effect of ethanol may be implicated in the development of muscle weakness with alcohol consumption. (+info)
Histamine-induced Ca2+ oscillations in a human endothelial cell line depend on transmembrane ion flux, ryanodine receptors and endoplasmic reticulum Ca2+-ATPase.
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Using single cell microfluorometry to monitor changes in bulk Ca2+ concentration ([Ca2+]bulk) and the whole-cell configuration of the patch clamp technique to measure K+ currents (voltage clamp) and membrane potential (current clamp), the mechanisms of histamine-induced Ca2+ oscillations in the umbilical vein endothelial cell-derived cell line EA.hy926 were studied. In single cells, histamine (10 microM) evoked sinusoidal Ca2+ oscillations in low extracellular Ca2+ concentrations ([Ca2+]o = 10-30 microM). In contrast, histamine did not initiate Ca2+ oscillations either in the absence of extracellular Ca2+ (10 microM EGTA) or in the presence of 2.5 mM extracellular Ca2+. Ca2+ oscillations were accompanied by rhythmic activation of Ca2+-activated K+ (KCa) channels and membrane hyperpolarization of 18.1 +/- 3.9 mV. Hence, cell depolarization with 70 mM extracellular K+ or the inhibition of non-selective cation channels (NSCCs) and KCa channels by 10 microM Loe 908 and 10 mM tetrabutylammonium prevented histamine-evoked Ca2+ oscillations. Preventing Na+-Ca2+ exchange (NCX) by 10 microM 2', 4'-dichlorobenzamil, or removal of extracellular Na+, abolished histamine-induced Ca2+ oscillations. Lowering the extracellular Na+ concentration and thus promoting the reversed mode of NCX (3Na+ out and 1Ca2+ in) increased the amplitude and frequency of histamine-induced Ca2+ oscillations by 25 and 13 %, respectively. Hence, in the absence of extracellular Ca2+, 10 microM histamine induced an elevation of intracellular Na+ concentration in certain subplasmalemmal domains. The inhibitor of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) 2,5-di-tert-butyl-1, 4-benzo-hydroquinone (15 microM) prevented histamine-induced Ca2+ oscillations. In addition, blockage of ryanodine-sensitive Ca2+ release (RsCR) by 25 microM ryanodine blunted Ca2+ oscillations. In endothelial cells that were treated for 16 h with 10 microM nocodazole to collapse the superficial endoplasmic reticulum (sER), no histamine-induced Ca2+ oscillations were found. We conclude that in low [Ca2+]o conditions histamine-induced Ca2+ oscillations depend on transmembrane Na+ loading through NSCCs that leads to Ca2+ entry via NCX. Cation influx is controlled by KCa channel activity that triggers membrane hyperpolarization and, thus, provides the driving force for cation influx. Hence, the Ca2+ entering needs to be sequestrated via SERCA into sER to become released by RsCR to evoke Ca2+ spiking. These data further support our previous work on localized Ca2+ signalling as a key phenomenon in endothelial Ca2+ homeostasis. (+info)
Effect of contractile activity on protein turnover in skeletal muscle mitochondrial subfractions.
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To determine the role of intramitochondrial protein synthesis (PS) and degradation (PD) in contractile activity-induced mitochondrial biogenesis, we evaluated rates of [(35)S]methionine incorporation into protein in isolated rat muscle subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria. Rates of PS ranged from 47 to 125% greater (P < 0.05) in IMF compared with SS mitochondria. Intense, acute in situ contractile activity (10 Hz, 5 min) of fast-twitch gastrocnemius muscle resulted in a 50% decrease in PS (P < 0.05) in SS but not IMF mitochondria. Recovery, or continued contractile activity (55 min), reestablished PS in SS mitochondria. In contrast, PS was not affected in either SS or IMF mitochondria after prolonged (60-min) contractile activity in the presence or absence of a recovery period. PD was not influenced by 5 min of contractile activity in the presence or absence of recovery but was reduced after 60 min of contractions followed by recovery. Chronic stimulation (10 Hz, 3 h/day, 14 days) increased muscle cytochrome-c oxidase activity by 2.2-fold but reduced PS in IMF mitochondria by 29% (P < 0.05; n = 4). PS in SS mitochondria and PD in both subfractions were not changed by chronic stimulation. Thus acute contractile activity exerts differential effects on protein turnover in IMF and SS mitochondria, and it appears that intramitochondrial PS does not limit the extent of chronic contractile activity-induced mitochondrial biogenesis. (+info)