Mary Lee S. Ledbetter

Control of protein synthesis in human fibroblasts by intracellular potassium

Abstract Intracellular potassium ion (K+) in cultured human fibroblasts (HF cells) was maintained at reduced steady-state levels by incubating cells in various ouabain concentrations. Small decreases in cell K+ had no effect on protein synthesis and cell growth, but when cell K+ fell below 60–80% of control levels, the rate of protein synthesis decreased in proportion to further reductions in K+. DNA synthesis was also inhibited, presumably because of its dependence on protein synthesis. On the other hand, RNA synthesis remained uninhibited over a wide range of K+ concentrations, an effect characteristic of many specific inhibitors of protein synthesis. In ouabain-treated cells neither levels of ATP nor transport of amino acids was limiting for protein synthesis. Loss of activity of messenger or other species of RNA was not responsible for inhibition of protein synthesis, since in the presence of actinomycin D, the rate of protein synthesis could be decreased or increased solely by adjusting cell K+. Release from ouabain inhibition restored K+ levels, macromolecular synthesis, and cell growth, but there was no resulting synchrony of cell division. In cell populations partially synchronized by serum starvation and refeeding protein synthesis was sensitive to reduction in K+ levels throughout the cell cycle. Our quantitative results show that cell K+ levels, when sufficiently reduced, can determine the rate of protein synthesis and hence the rate of cell growth.

Interference of ouabain in cell-to-cell coupling☆

Abstract When mouse cells are co-cultured with human cells in medium containing 1 μM ouabain, the steady-state culture content of 86Rb+ (a tracer for K+) is increased over that expected from the contents of the two types cultured separately. This increase is due to K+ transfer, presumably through gap junctions. Rescue of the human cells, which are sensitive to 1 μM ouabain, by K+ transfer from the mouse cells, which are not, may be expressed quantitatively by a function, index of cooperation, whose value for this combination of cells is usually 0.4–0.6. (In the absence of coupling, index of cooperation equals 0; theoretical maximum equals 1.0.) We now report that when the ouabain concentration was raised above 1 μM, the index of cooperation was correspondingly reduced. Similar observations were made on four different combinations of cells differing in sensitivity to ouabain but all capable of junctional communication under other conditions. The concentration of ouabain needed to block cooperation completely caused no detectable change in the steady-state K+ content of the donor cell, and was unaffected by substitution of a more ouabain-resistant donor or by a two-fold change in the proportion of donor and recipient cells in the co-culture. In confirmation, we observed that transfer of tritium-labeled nucleotides between cells was also blocked by treatment with ouabain. We interpret these results as indicating that ouabain interferes with functional communication, perhaps because of a local concentration of sensitive pump sites in junctional regions of the membrane, or because of inhibitory factors such as increased intracellular free Ca2+, that might result directly or indirectly from ouabain binding to the Na+,K+-ATPase.

Ouabain inhibition of cell communication may be associated with mobilization of cell calcium

To determine the mechanism of inhibition of communication among cultured animal cells by ouabain, we tested whether that inhibition could be related to changes in cell calcium. Human fibroblasts (HF) and mouse 3T3 cells showed increased exchangeable 45Ca2+ after ouabain treatment at concentrations sufficient to inhibit communication. HF cells also showed time- and concentration-dependent elevations in calcium using fura-2 fluorescence, but 3T3 cells did not. Treatment of HF cells with amiloride blocked the elevation of calcium, as well as the inhibition of cooperation between HF and 3T3 cells by ouabain. These data, together with the lack of effect of reduced extracellular calcium or of the channel-blocker verapamil, are consistent with a model in which ouabain treatment results in an amiloride-sensitive sodium influx, which then leads to elevated cytoplasmic free calcium. That, in turn, modulates communication through gap junctions.

Discipline-Based Education Research: Preaching to Converts Who Are Learning to Sing in the Choir

Scientists are becoming increasingly aware of the need to approach their teaching with the same expectations for evidence that they use in their science. They are coming to recognize that, just as in science research, research on undergraduate science education has a literature as well as standards for practice. I (M.L.L.) am a recent convert to this way of thinking. I taught for 30 years using mostly a lecture style and believed myself quite revolutionary when I used projected images to make cell biological points in class. I now teach using the results of discipline-based education research (DBER) and do my best to use instructional strategies that are demonstrated to improve student learning.