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The subcellular distribution of calcium in dermal melanocytes of Xenopus laevis and Poecilia reticulata has been analysed. Using two cytochemical methods, phosphate precipitation and a combined oxalate-pyroantimonate technique, electron energy-loss spectroscopy and electron spectroscopic imaging have been applied for elemental analysis. Both precipitation techniques revealed a high calcium content in the melanosomes of both species. Calcium was also located in the vicinity of collagen fibrils and in the plasma membrane.

Volume/osmotic pressure relationships were compared in Xenopus epidermal cells treated with the calcium antagonist lanthanum and in control cells. In lanthanum-treated cells the volume was found to be proportional to the reciprocal of the osmotic pressure in the hypotonic range, but not in the control cells. This difference is interpreted as being caused by contractions of the actomyosin system in control cells, which require external calcium. On the basis of the different slopes of the measurements under these two conditions the contractile force of the cells in isotonic conditions was calculated to be 0.96 x 10(5)Nm-2. The observation that in hypotonic media the cell body is enlarged in the direction of movement provides evidence that the cell body/lamella transition region is the part of the cell that contracts most during locomotion. By analysing cell speed in media with varying osmotic pressures, the relationship between internal pressure and cell locomotion was revealed.

Sequestration of iontophoretically injected Ca2+ by monolayer culture cells (primary Xenopus laevis Tadpole Heart cells, XTH P, and an established cell line, XTH 2) is investigated. Injections are made at different velocities by changing the influx current. On Ca2+ injection the entire ER desintegrates, and near to the tip of the injecting pipette microtubules depolymerize. The time required to attain cell death is taken as the parameter indicating an overload of cellular Ca2+ sequestration capability. Three different Ca2+ transport kinetics are found: at Ca2+ flux rates of up to 20 X 10(-15) mol X s-1 (condition I) cells can tolerate long injection periods before they die; at flux rates from 20 to 40 X 10(-15) mol X s-1 (condition II) the injection time before cell death remains constant. Flux rates exceeding 40 X 10(-15) mol X s-1 decrease cellular Ca2+ sequestration capability to a minimum. These observations support the assumption of two Ca2+ sequestrating mechanisms: one of high affinity, but with low capacity (less than = 5 X 10(-15) mol X s-1) the other with low affinity for Ca2+ and a high capacity (10 to 40 X 10(-15) mol X s-1) for Ca2+ accumulation. Both mechanisms are saturable. As the Ca2+ sequestration velocity remains approximately constant in condition II, the capacity of the second mechanism seems to grow with increasing Ca2+ influx. The highly affin Ca2+ compartment is the ER, mitochondria form the less affin system. XTH 2 differ from primary cells by possessing a 5 to 8 fold higher Ca2+ sequestration capacity, whereas sequestration velocity is equal in both cell types.

In XTH-2 cells, a line derived from tadpole heart endothelia, changes of the F-actin pattern have been followed after injection of calcium and after permeabilization of the cells to calcium using the ionophore A 23187 and an external pCa of 6.0. On the dorsal surface numerous microvilli appear, the stress fibres show an interrupted pattern and F-actin accumulates at the cell periphery. These reactions are interpreted to result from contractions. Finally most of the F-actin becomes depolymerized (in about 30 min). In addition actin containing accumulations are developed in the perinuclear area. In G1 cells stress fibres are less sensitive to enhanced calcium, they persist for more than 30 min in pCa 6.0. Taking into account a relatively high cytosolic calcium concentration in many malignantly transformed cells, their more rounded appearance and lack of an ordered array of actin fibrils could be closely related phenomena.

The role of calcium in the induction of locomotion, control of direction of locomotion, and modulation of shape of epithelial cells derived from Xenopus laevis tadpole epidermis is investigated. Local influx of calcium is achieved by electrophoretic release of small amounts of calcium from a micropipette (tip diameter 0.1-0.5 micron) closely apposed to the cell body or lamella. The cells are made permeable for calcium by calcium ionophore A23187, and they are kept in Ca++-free, Mg++-rich EGTA Ringer. Another method used to induce Ca++ influx is local application of A23187 while cells move in normal culture medium. Influx of Ca++ into the lamella induces a localised increase in thickness and enlargement of the lamella. Stationary cells become active and show movement in the direction of the Ca++ gradient. Fried-egg-shaped cells tend to acquire a semicircular shape and start moving. Moving cells change the direction of their locomotion, following the direction of Ca++ release. Influx of Ca++ in the cell body region induces its contraction concomitant with an increase in lamellar area. These observations suggest the presence of two different Ca++-sensitive components: an actomyosin meshwork in the cell body and an actin gel in the lamella. Influx of Ca++ induces contraction of actomyosin and solation of actin gel. Interaction of these two systems would explain modulation of shape and generation of locomotion in epithelial cells.

Endothelial cells derived from Xenopus tadpole hearts (XTH-2: Schlage et al. 1981) have been permeabilized for calcium using the ionophore A 23187. A Ca2+-concentration of 1 microM causes a graded disassembly of microtubules (mts): with increasing periods of incubation mts disintegrate and then depolymerize completely. The frequency of the different stages of depolymerization depends on the length of the Ca2+ incubation period, even after 60 min incubation time some mts remain. The stage of depolymerization of mts in a cell is correlated with its cell cycle phase showing the highest stability during S phase.

Dependence of locomotion of Xenopus laevis epidermal cells on calcium influx from the external medium was investigated. Inhibition of Ca2+ influx by 2 mM La3+ or 4 mM Tb3+ in the culture medium causes an immediate stop to locomotion and a loss of motion at the outer margin of the lamella; microcolliculi disappear and the entire lamella becomes flat and very thin. The cell body region enlarges by spreading into the lamella to an extent approximately coincident with the distribution of myosin. The increase in thickness of this area is the result. The cytoskeletal elements actin, alpha-actinin and myosin become homogeneously distributed throughout the cell and a great number of straight microtubules extend to the margin after 20 min in La3+-containing media. Prekeratin distribution does not change. Reduction of calcium concentration in the external medium by EGTA leads to cessation of cell locomotion. Sr2+ (1-4 mM) is also able to replace calcium for triggering locomotion. These findings point to a control of Ca2+-activated contractions of actomyosin by influx of external Ca2+. According to our model of cell locomotion [14] the contractions generate a hydrostatic pressure extending the lamella by flow of hyaloplasm towards the margin. Small swellings (microcolliculi) appearing thereby will be dislocated by a calcium-dependent sol-gel transformation in this area, which contains actin but not myosin.