Barium- and calcium-permeable channels open at negative membrane potentials in rat ventricular myocytes

Summary Ca²⁺- and Ba²⁺-permeable channel activity from adult rat ventricular myocytes, spontaneously appeared in the three single-channel recording configurations: cell-attached, and excised inside-out or outside-out membrane patches. Single-channel activity was recorded at steady-state applied membrane potentials including the entire range of physiologic values, and displayed no rundown in excised patches. This activity occurred in irregular bursts separated by quiescent periods of 5 to 20 min in cell-attached membrane patches, whereas in excised patch experiments, this period was reduced to 2 to 10 min. During activity, a variety of kinetic behaviors could be observed with more or less complex gating patterns. Three conductance levels: 22, 45 and 78 pS were routinely observed in the same excised membrane patch, sometimes combining to give a larger level. These channels were significantly permeable to divalent cations and showed little or no permeability to potassium or sodium ions. The inorganic blockers of voltage-gated Ca channels, cobalt (2mm), cadmium (0.5mm) or nickel (3mm), had no apparent effect on these spontaneous unitary currents carried by barium ions. Under 10^–5 m bay K 8644 or nitrendipine, the activity was clearly increased in about half of the tested excised inside-out membrane patches. Both dihydropyridines enhanced openings of the larger conductance level, which was only very occasionally seen under control conditions. When the single-channel activity became sustained under 5×10^–6 m Bay K 8644, it was possible to calculate the mean unitary current at different membrane potentials and show that the mean current value increased with membrane potential.     

Entrainment and phase shifting of the circadian rhythm of cell division by light in cultures of the achlorophyllous ZC mutant ofEuglena gracilis

The photosynthesis-deficient ZC mutant ofEuglena gracilis Klebs (strain Z) was cultured at 16°C on an aerated, magnetically stirred, mineral medium containing 0.1% ethanol (pH 7.0). Cell division could be entrained by a 12: 12 light: dark cycle (LD: 12, 12) or even by a one-pulse skeleton photoperiod (LD: 1,23) The rhythm free-ran in DD for at least 8 days with a circadian period (=25.5 h) in populations that had been previously entrained by LD. The freerunning rhythm could be phase-shifted by a single 1-h light pulse (3000 lx). The strong (Type 0) phase-response curve derived from the resetting effects of such signals given at different circadian times was similar to that for the photosynthetic wild-type strain. These results demonstrate that the presence of a functional chloroplast compartment is not necessary for the circadian clock to function inEuglena and suggest that phase resetting of the circadian clock by light occurs via a similar pathway in both photosynthetic and non-photosynthetic cell types.     

Alterations in Protective Enzymes Against Peroxidation in the Central and Peripheral Nervous System of Control and Dysmyelinating Mutant Mice

The activities of peroxide-detoxifying enzymes such as superoxide dismutase (SOD), glutathione peroxidase, glutathione reductase, and catalase were measured in the nervous system of neurological dysmyelinating mutants: quaking (Qk), shiverer (Shi), and trembler (Tr) mice. Cu/Zn-SOD activity was higher in the cerebellum of Qk and Shi mice (by 53% and 106%, respectively) in comparison with controls, but it was the same in the cerebellum of Tr mice and their corresponding controls. In contrast, there was no difference in the level of Cu/Zn-SOD activity in the cerebrum of Qk, Shi, and Tr mice and their respective controls. Mn-SOD activity was the same among all the mutants compared to control animals in both cerebrum and cerebellum. In Shi cerebellum, glutathione peroxidase and glutathione reductase activities were slightly decreased (a 21.6% and a 13.2% diminution, respectively), whereas catalase activity in cerebrum and cerebellum was the same among mutants and control mice. In the sciatic nerve from Tr mice, all the enzymatic activities were enhanced: sixfold increase for total SOD, and 2.4-fold, 3.5-fold, and 1.8-fold increase for glutathione peroxidase, glutathione reductase, and catalase, respectively.     

Effect of lysophosphatidylcholine on the NADH-ferricyanide reductase activity associated with the plasma membranes of corn roots

Plasma membranes from corn roots (Zea mays L.) were isolated by aqueous two-phase partitioning. A fraction enriched in a vanadate-sensitive ATPase showed characteristics of a plasma membrane ATPase. The sidedness of these vesicles was 89% right-side-out, as evaluated by the ATPase latency. A NADH-ferricyanide reductase was associated with these plasma membrane vesicles. The rate of ferricyanide reduction was 1.3 μmol · min⁻¹·mg⁻¹ protein and was strongly enhanced by the addition of lysophosphatidylcholine (LPC). The effect of this detergent on membrane solubilization and reductase activity was particularly studied. This type of detergent treatment revealed two pH optima (7.0 and 5.0) for the reductase activity, which exhibited biphasic kinetics in the absence or presence of the detergent. These data suggest that two or more reductases could be involved. In addition, membrane vesicle solubilization and determination of ATPase and reductase latency were simultanously studied. From these experiments, it is postulated that the reductase, which exhibits an optimum pH at 7.0 and is slightly stimulated by LPC, could be located on the external side of the plasmalemma. In contrast, the reductase at pH 5.0 strongly stimulated by the detergent treatment, is probably located on the internal side of the membrane, such as the catalytic site of ATPase. Finally, a possible direct action of LPC on the enzymes, is discussed.     

Involvement of the Dihydropyridine Receptor and Internal CaStores in Myoblast Fusion

The process of myoblast fusion during skeletal myogenesis is calcium regulated. Both dihydropyridine receptor and ryanodine receptor are already present on muscle precursors, at the prefusional stage, before they are required for excitation–contraction coupling. Previous pharmacological studies have shown the need for a special pool of Ca²⁺associated with the membrane for the fusion process to occur. We hypothesized that this pool of Ca²⁺is mobilized via a machinery similar to that involved in excitation–contraction coupling. The process of fusion in rat L6 muscle precursors was either totally or partially abolished in the presence of the L-type calcium channel inhibitors SR33557 and nifedipine (half inhibition towards 2 μM), respectively. The inhibition was reversible and dose-dependent. Drugs able to deplete internal calcium stores (caffeine, ryanodine, and thapsigargin) were also tested on the fusion. Both caffeine and thapsigargin drastically inhibited fusion whereas ryanodine had no effect. This suggests that fusion may be controlled by internal pools of Ca²⁺but that its regulation may be insensitive to ryanodine. We presumed that an early form of the ryanodine receptor may exist, with different pharmacological properties than the adult forms. Indeed, Western blot analysis of pre- and postfusional L6 cells demonstrated the presence, at the prefusional stage, of a transient form of the ryanodine receptor protein with an apparent molecular weight slightly different from those of the classical skeletal and cardiac forms. Taken together, these results support the hypothesis that the fusion process is driven by a mechanism involving both the dihydropyridine receptor (α1 subunit of the L-type Ca²⁺channel) and the internal stores of Ca²⁺. The machinery underlying this mechanism might consist of slightly different forms of the classic molecules that in adult muscle ensure excitation–contraction coupling. It remains to be seen, however, whether the mobilization of the internal pool of Ca²⁺is triggered by the type of mechanism already described in skeletal muscle.