Static and magic angle spinning (31)P NMR spectroscopy of two natural plasma membranes.
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Static and magic angle spinning (31)P NMR spectroscopy was used for the first time in natural plasma membranes from erythrocytes and skeletal muscle to study phospholipid arrangement and composition. Typical static powder-like spectra were obtained showing that phospholipids were in a bilayer arrangement. Magic angle spinning narrowed spectra into two components. The first one corresponded to phosphatidylcholine and the second one to the other phospholipids with intensities in agreement with the known phospholipid composition. These findings show that NMR data previously acquired using model membranes can be transposed to studies on phospholipids in their natural environment. (+info)
Annexin VI participates in the formation of a reversible, membrane-cytoskeleton complex in smooth muscle cells.
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The plasmalemma of smooth muscle cells is periodically banded. This arrangement ensures efficient transmission of contractile activity, via the firm, actin-anchoring regions, while the more elastic caveolae-containing "hinge" regions facilitate rapid cellular adaptation to changes in cell length. Since cellular mechanics are undoubtedly regulated by components of the membrane and cytoskeleton, we have investigated the potential role played by annexins (a family of phospholipid- and actin-binding, Ca(2+)-regulated proteins) in regulating sarcolemmal organization. Stimulation of smooth muscle cells elicited a relocation of annexin VI from the cytoplasm to the plasmalemma. In smooth, but not in striated muscle extracts, annexins II and VI coprecipitated with actomyosin and the caveolar fraction of the sarcolemma at elevated Ca(2+) concentrations. Recombination of actomyosin, annexins, and caveolar lipids in the presence of Ca(2+) led to formation of a structured precipitate. Participation of all 3 components was required, indicating that a Ca(2+)-dependent, cytoskeleton-membrane complex had been generated. This association, which occurred at physiological Ca(2+) concentrations, corroborates our biochemical fractionation and immunohistochemical findings and suggests that annexins play a role in regulating sarcolemmal organization during smooth muscle contraction. (+info)
S-T segment voltage during sequential coronary occlusions is an unreliable marker of preconditioning.
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During coronary angioplasty, a stair-step decrease in peak S-T segment elevation from the first to the second coronary occlusion has been assumed to indicate a preconditioning (PC) effect. This association was evaluated with myocardial electrograms in rabbits, which revealed that two sequential 5-min coronary occlusions resulted in a marked decrease in the area under the S-T segment voltage-time curve (P < 0.05) with no change during a third occlusion. Pretreatment with either 5-hydroxydecanoate, a mitochondrial ATP-sensitive potassium (K(ATP)) channel blocker, or anisomycin, an activator of stress-activated protein kinases, had no effect on the stair-step decline in the S-T segment voltage between the first two occlusions. HMR-1883, a potent closer of sarcolemmal K(ATP) channels, abolished changes in S-T segment elevation after brief coronary occlusions but had no effect on the infarct-sparing property of the two preconditioning 5-min occlusions. Interestingly, HMR-1883 blocked myocardial protection from diazoxide, raising doubt that the latter opens only mitochondrial channels. Therefore, myocardial protection and S-T segment changes during ischemia are dissociated. These data suggest that it is the mitochondrial K(ATP) channel that protects the myocardium, and it is the sarcolemmal channel that is responsible for changes in S-T elevation. Therefore, it cannot always be inferred that changes in S-T segment elevation reflect the state of myocardial protection. (+info)
Biochemical and functional evidences for a GLUT-4 homologous protein in avian skeletal muscle.
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The characteristics and modulation of glucose transport were investigated in skeletal muscles of 5-wk-old Muscovy ducklings (Cairina moschata). Glucose uptake by sarcolemmal vesicles isolated from gastrocnemius muscle followed typical Michaelis-Menten kinetics with a K(m) value (17 mM) similar to that described in equivalent mammalian preparations. Western blot analysis of duckling sarcolemma using antibodies directed against rat GLUT-4 transporter revealed an immunoreactive protein of similar molecular mass (45 kDa) to that present in rats. When ducklings were killed in the postabsorptive state, GLUT-4 homologous protein was located predominantly (80%) in intracellular membranes. Insulin stimulation of a perfused leg muscle preparation in vitro led to the translocation of GLUT-4 homologous proteins from intracellular pools to the sarcolemma, with a subsequent increase in glucose uptake by sarcolemmal vesicles and perfused muscles. Glucose transport was positively controlled by the metabolic needs of skeletal muscle as reflected by the increased glucose uptake of sarcolemmal vesicles isolated from cold-acclimated ducklings. Present results, therefore, demonstrate, for the first time in an avian species, the existence in skeletal muscle of a glucose transporter showing molecular and functional homologies with the mammalian GLUT-4 transporter. (+info)
Factors affecting membrane permeability and ionic homeostasis in the cold-submerged frog.
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Frogs (Rana temporaria) were submerged at 3 degrees C in either normoxic (P(O2)=155 mmHg, P(O2)=20 kPa) or hypoxic (P(O2)=60 mmHg; P(O2)=8 kPa) water for up to 16 weeks, and denied air access, to mimic the conditions of an ice-covered pond during the winter. The activity of the skeletal muscle Na(+)/K(+) pump over the first 2 months of hibernation, measured by ouabain-inhibitable (22)Na(+) efflux, was reduced by 30 % during normoxia and by up to 50 % during hypoxia. The reduction in Na(+)/K(+) pump activity was accompanied by reductions in passive (22)Na(+) influx and (86)Rb(+) efflux (effectively K(+) efflux) across the sarcolemma. This may be due to a decreased Na(+) permeability of the sarcolemma and a 75 % reduction in K(+) leak mediated by ATP-sensitive K(+) channels ('K(ATP)' channels). The lowered rates of (22)Na(+) and (86)Rb(+) flux are coincident with lowered transmembrane ion gradients for [Na(+)] and [K(+)], which may also lower Na(+)/K(+) pump activity. The dilution of extracellular [Na(+)] and intracellular [K(+)] may be partially explained by increased water retention by the whole animal, although measurements of skeletal muscle fluid compartments using (3)H-labelled inulin suggested that the reduced ion gradients represented a new steady state for skeletal muscle. Conversely, intracellular ion homeostasis within ventricular muscle was maintained at pre-submergence levels, despite a significant increase in tissue water content, with the exception of the hypoxic frogs following 4 months of submergence. Both ventricular muscles and skeletal muscles maintained resting membrane potential at pre-submergence levels throughout the entire period of hibernation. The ability of the skeletal muscle to maintain its resting membrane potential, coincident with decreased Na(+)/K(+) pump activity and lowered membrane permeability, provided evidence of functional channel arrest as an energy-sparing strategy during hibernation in the cold-submerged frog. (+info)
Hyperaldosteronemia in rabbits inhibits the cardiac sarcolemmal Na(+)-K(+) pump.
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Aldosterone upregulates the Na(+)-K(+) pump in kidney and colon, classical target organs for the hormone. An effect on pump function in the heart is not firmly established. Because the myocardium contains mineralocorticoid receptors, we examined whether aldosterone has an effect on Na(+)-K(+) pump function in cardiac myocytes. Myocytes were isolated from rabbits given aldosterone via osmotic minipumps and from controls. Electrogenic Na(+)-K(+) pump current, arising from the 3:2 Na(+):K(+) exchange ratio, was measured in single myocytes using the whole-cell patch clamp technique. Treatment with aldosterone induced a decrease in pump current measured when myocytes were dialyzed with patch pipette solution containing Na(+) in a concentration of 10 mmol/L, whereas there was no effect measured when the solution contained 80 mmol/L Na(+). Aldosterone had no effect on myocardial Na(+)-K(+) pump concentration evaluated by vanadate-facilitated [(3)H]ouabain binding or by K(+)-dependent paranitrophenylphosphatase activity in crude homogenates. Aldosterone induced an increase in intracellular Na(+) activity. The aldosterone-induced decrease in pump current and increased intracellular Na(+) were prevented by cotreatment with the mineralocorticoid receptor antagonist spironolactone. Our results indicate that hyperaldosteronemia decreases the apparent Na(+) affinity of the Na(+)-K(+) pump, whereas it has no effect on maximal pump capacity. (+info)
Modulation of CICR has no maintained effect on systolic Ca2+: simultaneous measurements of sarcoplasmic reticulum and sarcolemmal Ca2+ fluxes in rat ventricular myocytes.
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1. The effects of modulating Ca2+-induced Ca2+ release (CICR) in single cardiac myocytes were investigated using low concentrations of caffeine (< 500 microM) in reduced external Ca2+ (0.5 mM). Caffeine produced a transient potentiation of systolic [Ca2+]i (to 800 % of control) which decayed back to control levels. 2. Caffeine decreased the steady-state sarcoplasmic reticulum (SR) Ca2+ content. As the concentration of caffeine was increased, both the potentiation of the systolic Ca2+ transient and the decrease in SR Ca2+ content were increased. At higher concentrations, the potentiating effect decayed more rapidly but the rate of recovery on removal of caffeine was unaffected. 3. A simple model in which caffeine produces a fixed increase in the fraction of SR Ca2+ which is released could account qualitatively but not quantitatively for the above results. 4. The changes in total [Ca2+] during systole were obtained using measurements of the intracellular Ca2+ buffering power. Caffeine initially increased the fractional release of SR Ca2+. This was followed by a decrease to a level greater than that under control conditions. The fraction of systolic Ca2+ which was pumped out of the cell increased abruptly upon caffeine application but then recovered back to control levels. The increase in fractional loss is due to the fact that, as the cytoplasmic buffers become saturated, a given increase in systolic total [Ca2+] produces a larger increase in free [Ca2+] and thence of Ca2+ efflux. 5. These results confirm that modulation of the ryanodine receptor has no maintained effect on systolic Ca2+ and show the interdependence of SR Ca2+ content, cytoplasmic Ca2+ buffering and sarcolemmal Ca2+ fluxes. Such analysis is important for understanding the cellular basis of inotropic interventions in cardiac muscle. (+info)
Influence of tension time on muscle fiber sarcolemmal injury in rat diaphragm.
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We hypothesized that the amount of sarcolemmal injury is directly related to the total tension time (TT(tot)), calculated as mean tension x total stimulation time. Diaphragm strips from Sprague-Dawley rats were superfused at optimal muscle length with Krebs containing procion orange to identify sarcolemmal injury. TT(tot) was induced by stimulation with 100 Hz for 3 min at duty cycles of 0.02, 0.15, 0.3, and 0.6, or with continuous contractions at 0.2, 0.4, 0.6, and 1.0 of maximal tension. A significant positive correlation between TT(tot) and the percentage of fibers with injured sarcolemma (r(2) = 0.63, P < 0.05) is seen. Stimulation (at 100 Hz, duty cycle = 1) resulted in fast fatigue with low injury, likely caused by altered membrane conductivity. Stimulations inducing the largest injury are those showing progressive force loss and high TT(tot), where injury may be due to activation of membrane degradative enzymes. The maximal tension measured at 20 min poststimulation was inversely related to the number of fibers injured, suggesting loss of force is caused by cellular injury. (+info)