No abstract

We have measured the distribution of intracellular calcium concentration in isolated single muscle fibres from Xenopus laevis using the fluorescent calcium indicator fura-2 with digital imaging fluorescence microscopy. Under control conditions, resting and tetanic calcium were uniform throughout a fibre. When fatigue was produced using a prolonged, high-frequency tetanus, the distribution of calcium within muscle fibres became non-uniform, with greater levels near the outer parts of a fibre than near the centre. This non-uniform distribution of calcium was rapidly abolished by lowering the stimulation frequency. When fatigue was produced using a series of repeated intermittent tetani, tetanic calcium showed an initial small increase, followed by a decrease as stimulation was continued. The distribution of calcium remained uniform under these conditions. Calcium distribution was also uniform during recovery from intermittent tetanic stimulation. Although fibres varied considerably in their fatigue resistance, the time for tension to fall to 50% was correlated with the reduction in tetanic calcium seen at this time. These results indicate that there are at least two patterns of reduced calcium release that can contribute to the development of fatigue. The appearance of a calcium gradient is consistent with impaired t-tubular conduction, while a uniform reduction of calcium is likely to be due to the action of metabolic factors on systems controlling calcium homeostasis within the cell.

During terminal differentiation, keratinocytes lose the ability to divide. One indicator of responsiveness to certain growth factors is a transient rise in the intracellular concentration of free calcium ions ([Ca2+]i). The aim of our experiments was to discover whether or not terminally differentiating keratinocytes have lost the ability to exhibit an increase in [Ca2+]i in response to factors that stimulate [3H]thymidine incorporation and increase [Ca2+]i in undifferentiated keratinocytes. [Ca2+]i was measured with the calcium indicator dye FURA-2 and by a ratio imaging method. Expression of involucrin, a precursor of the keratinocyte cornified envelope, was used as a marker of terminal differentiation. Measurements were made on stratified colonies of cells grown in standard medium (containing 1.8 mM calcium ions) and on cell monolayers in low calcium medium (0.1 mM). Treatment of serum-starved monolayers with substance P, bombesin or complete growth medium containing 10% fetal calf serum resulted in increased [3H]thymidine incorporation. A switch from low calcium to standard medium also stimulated [3H]thymidine incorporation whether or not the cells had been serum-starved. In each experiment some cells showed an increase in [Ca2+]i while others did not. However, the heterogeneity in the [Ca2+]i response did not reflect the terminal differentiation status of individual cells: both involucrin-positive and -negative cells were found in the responding and nonresponding populations. Involucrin-positive and -negative areas of stratified cultures also underwent a transient increase in [Ca2+]i in response to serum-containing medium. Our data therefore indicate that both proliferating (involucrin-negative) and post-mitotic, terminally differentiating (involucrin-positive) keratinocytes can respond to mitogenic stimuli by an increase in [Ca2+]i.(ABSTRACT TRUNCATED AT 250 WORDS)