Kiyota Goshima

Ionic mechanism of morphological changes of cultured myocardial cells on successive incubation in media without and with Ca2

Abstract (1) Incubation of cultured mouse myocardial cells in medium containing Ca 2+ after brief pre-incubation in Ca 2+ -free medium caused morphological changes, such as full contraction of myofibrils and balloon formation of the cell membrane (“Ca 2+ paradox phenomenon”). (2) When myocardial cells were pre-incubated in Ca 2+ -free medium, and then incubated in medium containing various concentrations of Ca 2+ , both the percentage of cells showing morphological changes and the rate of Ca 2+ uptake increased with increase in the Ca 2+ concentration. (3) Pre-incubation in medium containing 10 −7 m Ca 2+ or less was necessary for induction of both morphological changes and excess uptake of Ca 2+ during incubation in medium containing Ca 2+ . (4) When myocardial cells were pre-incubated in Ca 2+ -free medium containing various concentrations of Na + , and then incubated in medium containing Ca 2+ , both the percentage of cells showing morphological changes and the rate of Ca 2+ uptake increased with increase in the Na + concentration in the pre-incubation medium. (5) Various treatments that inhibited excess uptake of Ca 2+ by the cells inhibited the genesis of morphological changes. Thus the observed morphological changes of myocardial cells were due to excess uptake of Ca 2+ by the myocardial cells. (6) When myocardial cells were incubated in medium containing various concentrations of Ca 2+ and a fixed concentration of Na + , the intracellular concentration of Na + increased with decrease in the Ca 2+ concentration of the medium. Cells that had been preloaded with a higher concentration of Na + took up the Ca 2+ faster. (7) Conditions that inhibited Na + uptake by the cells during pre-incubation without Ca 2+ inhibited Ca 2+ uptake by the cells during subsequent incubation with Ca 2+ . These results suggest that Ca 2+ uptake by myocardial cells during incubation in medium with Ca 2+ depends upon the intracellular Na + concentration.

Kinetic studies on sodium-dependent calcium uptake by myocardial cells and neuroblastoma cells in culture

Kinetic analyses were made on intracellular Na+-dependent Ca2+ uptake by myocardial cells and neuroblastoma cells (N-18 strain) in culture. Cells loaded with various concentrations of Na+ could be prepared by incubating them in Ca2+-free medium containing various concentrations of Na+. Cells pre-loaded with various concentrations of Na+ were incubated in medium containing Ca2+ and 45Ca. The resulting 45Ca uptake by the two types of cell depended greatly on the initial intracellular concentrations of Na+. Lineweaver-Burk plots of the initial rate of Ca2+ uptake against the external concentration of Ca2+ fitted well to straight lines obtained by linear regression (r greater than 0.95). This result shows that Ca2+ uptake by the two types of cell was achieved by a carrier-mediated transport system. This Na+-dependent Ca2+ uptake was accompanied by Na+ release and the ratio of Na+ release to Ca2+ uptake was close to 3 : 1. A comparison of the kinetic data between myocardial cells and N-18 cells suggested that N-18 cells possess a carrier showing the same properties as that of myocardial cells, i.e.: (1) a similar dependency on the intracellular concentration of Na+; (2) the coincidence of the apparent Michaelis constants for Ca2+ (0.1 mM); (3) the similarities of the Ki values for Co2+, Sr2+ and Mg2+ (Co2+ less than Sr2+ less than Mg2+) and (4) a similar dependency on pH. However, the maximal initial rate, V, of N-18 cells was about 1/100 that of myocardial cells. The rate of Na+-dependent Ca2+ uptake by non-excitable cells was much lower than that by myocardial cells.

Comparison of kinetic characteristics of Na+ -Ca2+ exchange in sarcolemma vesicles and cultured cells from chick heart

The kinetic characteristics of Na+-Ca2+ exchange in isolated sarcolemma vesicles from new-borne chick heart, which contain about 70% of right-side-out vesicles, were compared with those of cultured embryonic chick heart cells. Na+-Ca2+ exchange was monitored as Nai-dependent Ca2+ uptake. Increase in the internal concentration of Na+ ([Na+]i) in these two preparations caused increase in both the initial rate and the saturation-level of Ca2+ uptake. Plots of the rate of Ca2+ uptake against [Na+]i showed similar saturation-kinetics in these two preparations. The apparent Michaelis constant (Km) (0.35 mM) for Ca2+ uptake by the intact cells was much higher than that (0.031 mM) for Ca2+ uptake by the vesicles. The degree of inhibition by Mg2+ was also higher in the cells than in the vesicles. Some possible reasons (age of the chicks used, membrane potential, etc.), for these differences were examined and are discussed.

Partial purification of Na+−Ca2+ antiporter from plasma membrane of chick heart

To study Na+-Ca2+ exchange, proteins of membrane vesicles from chick hearts were solubilized with cholate in the presence of phospholipids and the cholate extract was treated with pronase. These purified proteoliposomes, reconstituted by subsequent dilution and centrifugation to eliminate the cholate, catalyzed Ca2+ uptake depending on the intraliposomal Na+ (Nai+) concentration. The maximal amount of Ca2+ accumulating in the liposomes was 140 nmol/mg protein and the initial rates of Nai+-dependent Ca2+ uptake were routinely 20 to 40 nmol/mg per 3 s at 25 degrees C, but only 2 to 4 nmol/mg per 3 s for the crude proteoliposomes from the cholate extract not treated with pronase. Thus the pronase treatment resulted in 10-fold purification. Nai+-dependent Ca2+ uptake by purified proteoliposomes was 30- to 50-fold higher than that by the initial membrane vesicles. The fundamental properties of Nai+-dependent Ca2+ uptake in purified proteoliposomes such as Km for Ca2+, the sensitivity for Na+ and pH dependency, were nearly equal to those in membrane vesicles and crude proteoliposomes. Thus, pronase treatment was very useful for obtaining reconstituted liposomes containing highly enriched Na+-Ca2+ antiporters which were functionally intact.

Involvement of an Na+Ca2+ exchange system in genesis of ouabain-induced arrhythmias of cultured myocardial cells

Abstract The changes of sarcomere length of cultured myocardial cells during genesis of ouabain-induced fibrillatory beating were measured, and it was found that the sarcomere continued to be in the shortened state throughout the fibrillatory beating. Addition of ouabain caused gradual increases of both the Na content of myocardial cells and the rate of Ca uptake by the cells, and fibrillatory beating appeared to develop when the Na content and the rate of Ca uptake exceeded the normal levels by about 1.5 times and 2.0 times, respectively. Myocardial cells loaded with various concentrations of Na + could be prepared by incubating the cells in Ca 2+ -free medium containing various concentrations of Na + , and it was found that a slight increase in the intracellular Na content from the physiological concentration caused an appreciable increase in Ca uptake by the cells. This Ca uptake was achieved by a carrier-mediated Na + Ca 2+ exchange system and the stoichiometry of Na + : Ca 2+ exchange was greater than 2 Na + :1 Ca 2+ . From these observations the possible involvement of an Na + Ca 2+ exchange system in genesis of ouabain-induced arrhythmias of cultured myocardial cells is discussed.

The role of extracellular calcium ions in HVJ (Sendai virus)-induced cell fusion.

The biochemical and biophysical roles of extracellular calcium ions in HVJ (Sendai virus)-induced cell fusion were studied. (1) Various kinds of cell, such as Ehrlich ascites tumor cells, mouse melanoma cells (B16-CW1 cells) and human epidermoid carcinoma cells (KB cells), could fuse in Ca2+-free medium containing a cheletor, glycoletherdiaminetetraacetic acid, in the same way as in Ca2+-containing medium. (2) The ATP content in Ehrlich ascites tumor cells decreased rapidly when the cells were treated with the virus in Ca2+-free medium but not in Ca2+-containing medium. (3) Intracellular adenine nucleotides leaked out into the reaction medium when the cells were treated with the virus in Ca2+-free medium but not in Ca2+-containing medium. (4) On addition of the virus, O2 consumption of Ehrlich ascites tumor cells decreased in Ca2+-free medium, but not in Ca2+-containing medium. (5) HVJ (Sendai virus) did not affect production of lactate by Ehrlich ascites tumor cells in both Ca2+-free medium and Ca2+-containing medium. These observations suggest that the role of extracellular Ca2+ in virus-induced cell fusion is to maintain the ATP and other intracellular metabolite contents at normal levels instead of triggering the fusion reaction itself.

Arrhythmogenic action of maitotoxin in guinea-pig and rat cardiac muscle

Abstract Maitotoxin, the most potent marine toxin known, produced arrhythmogenic effects on guinea-pig isolated atria. The time to onset of arrhythmia induced by maitotoxin (above 2 ng/ml) was shortened in a concentration-dependent manner. Maitotoxin (0.01-1 ng/ml) caused various arrhythmic movements in both cultured and isolated rat myocardial cells. The maitotoxin-induced tachyarrhythmia was abolished by treatment of cultured cardiac myocytes with verapamil (0.1 μM). Maitotoxin (1 ng/ml) increased the 45 Ca uptake of cultured cardiac myocytes and the cytoplasmic free Ca 2+ concentration of isolated myocardial cells. Maitotoxin (up to 10 ng/ml) had no effect on the enzyme activity of cardiac Na + ,K + -ATPase, c-AMP phosphodiesterase and sarcoplasmic reticulum Ca 2+ -ATPase. The electrophysiological effects of maitotoxin on isolated guinea-pig cardiac myocytes were examined by means of whole-cell patch-clamp techniques. Maitotoxin did not increase the transient Ca channel current but instead produced a steadily flowing current, which was abolished by Cd 2+ (1 mM). The voltage dependence curve for the maitotoxin-induced current was almost linear and the amplitude of this current increased as the external Ca 2+ concentration was elevated from 0.45 to 1.8 mM. These results suggest that maitotoxin directly increases a current flowing through the cardiac muscle membrane which is carried by Ca 2+ . This could elevate the intracellular free Ca 2+ concentration to result in a Ca-overloaded state and thus induce arrhythmogenic effects.

Selective killing of fibroblast-like cells in cultures of mouse heart cells by treatment with a Ca ionophore, A23187

Abstract Myocardial cells uncontaminated by fibroblast-like cells could be obtained by treating mouse heart cell cultures with a Ca ionophore, A23187. When trypsin-dissociated mouse heart cells were cultivated for 1 day or more on Petri dishes, both myocardial and fibroblast-like cells were detected on the dish under a phase contrast microscope. However, when the cultured heart cells were treated for a short period (e.g. 10 min) at 25 °C with A23187, only fibroblast-like cells exhibited morphological degeneration, such as nuclear pycnosis, cytoplasmic condensation and bleb- and balloon-formation. The myocardial cells maintained their normal morphology during the treatment; transient irregular beating was observed, but disappeared after removal of A23187. Thus treatment of heart cell cultures with A23187 can yield a large amount of myocardial cells without contamination by fibroblast-like cells. The possible mechanism for this selective effect is discussed.

Inhibition of ouabain-induced arrhythmias of ouabain-sensitive myocardial cells (quail) by contact with ouabain-resistant cells (mouse) and its mechanism

Abstract (1) Using cultured quail and mouse myocardial cells, studies were made on the ouabain sensitivity of ouabain-sensitive myocardial cells (quail) in contact with ouabain-resistant myocardial cells (mouse) in mosaic cell-sheets, and the role of Na diffusion from quail myocardial cells to neighbouring mouse myocardial cells in prevention of ouabain-induced arrhythmias of the mosaic cell-sheets. (2) Addition of ouabain caused gradual increase in the intracellular Na content of cultured quail and mouse myocardial cells, and arrhythmias appeared to develop when the Na content exceeded about 1.5 times the normal level (20 m m Na). The ouabain concentrations necessary for induction of increase in Na content and genesis of arrhythmias were high for mouse myocardial cells and low for quail myocardial cells. (3) When the Na contents of quail and mouse myocardial cells had been elevated by treatment with ouabain, they gradually decreased on washing-out the ouabain, and when they became less than 1.3 times the normal level, the arrhythmias disappeared and normal beating re-started. (4) When quail myocardial cells (ouabain-sensitive) in contact with mouse myocardial cells (ouabain-resistant) formed confluent mosaic cell-sheets, these sheets became progressively more resistant to ouabain-induced arrhythmias with increase in the proportion of mouse myocardial cells in the sheets. (5) Ouabain-induced increase in the Na content of the mosaic cell-sheets was less than expected from the increases in sheets entirely composed of each type. (6) Fused cells containing one mouse and one quail myocardial cell nucleus and cell-pairs composed of one mouse and one quail myocardial cells had similar ouabain sensitivity for genesis of arrhythmias. (7) Contact of quail myocardial cells with ouabain-resistant non-excitable cells (3T3 cells) which are able to form functional contact (mediate synchronous beating of myocardial cells or form electrical coupling) with myocardial cells inhibited the ouabain-induced arrhythmias of the quail cells. (8) These observations suggest that the mechanism of prevention of ouabain-induced arrhythmias of ouabain-sensitive myocardial cells by contact with ouabain-resistant cells is as follows: When a suitable concentration of ouabain is added to mosaic cell-sheets composed of ouabain-sensitive myocardial cells and ouabain-resistant cells, the ouabain inhibits the Na pump of only the oubain-sensitive myocardial cells, thus the Na content of the oubain-sensitive myocardial cells increases. The Na ions are then transmitted to neighbouring ouabain-resistant cells, probably through low-resistance junctions (gap junctions), and pumped out through the functioning Na pump of these ouabain-resistant cells.

Beating activity of heterokaryons between myocardial and non-myocardial cells in culture

Cultured mouse myocardial cells grown as monolayers fused upon treatment with HVJ (Sendai virus). The myocardial cells also fused with quail myocardial cells, neuroblastoma cells and non-excitable cells, such as KB cells. The beating activity of these heterokaryons was studied in the present work. Heterokaryons composed of myocardial cells from different species maintained spontaneous beating activity for 2 days or more. Those of one myocardial and one neuroblastoma cell maintained the activity for 22-26 h, while those of one myocardial and one non-excitable cell, such as KB cell, lost the activity within 2-4 h after addition of HVJ. Heterokaryons that had stopped spontaneous beating did not contract on application of electrical-field stimulation. The ration of non-myocardial cells in the heterokaryons increased in inverse proportion to the decrease in beating activity of the heterokaryons. Study of the rapid disappearance of beating activity in heterokaryons composed of one myocardial and one KB cell showed that both excitability of the cell membrane and myofibril organization were rapidly lost.