Immunological detection of cell surface components related with aggregation of Chinese hamster and chick embryonic cells.

Previous studies suggested that Chinese hamster V79 cells possess two mechanisms for their mutual adhesion, Ca²⁺-dependent and Ca²⁺-independent ones. We could prepare cells with only the Ca²⁺-dependent mechanism intact by dispersing cell monolayers with trypsin (0.01%) containing Ca²⁺. In the present study, we found that cells dispersed with a very low concentration of trypsin (0.0001%) in the absence of Ca²⁺ retain only the Ca²⁺-independent mechanism intact. Fab fragments of antibodies directed against surface antigens of V79 cells inhibited the aggregation of V79 cells by the Ca²⁺-independent mechanism, but did not inhibit the aggregation of these cells by the Ca²⁺-dependent mechanism. These results suggest that the two mechanisms of cell adhesion are based on different cellular components. Molecules responsible for the Ca²⁺-independent adhesion mechanism are probably cell surface components, because they were released from cells by the treatment with 0.01% trypsin without losing their specific antigenicity. The presence of adhesion mechanisms similar to those in V79 cells was shown in neural retinal cells of chick embryos. It was assumed, therefore, that these mechanisms of cell adhesion are generally present among a variety of cell types.     

Cleavage stage mouse embryos share a common cell adhesion system with teratocarcinoma cells

We examined similarities in adhesive properties of mouse cleaving embryos at one- to eight-cell stages and of teratocarcinoma cells by aggregation studies. Teratocarcinoma cells and fibroblastic cells have a Ca²⁺-dependent cell-cell adhesion site (CDS), which is resistant to trypsin in the presence of Ca²⁺ but sensitive in the absence of Ca²⁺. When several embryos treated with trypsin in the presence of Ca²⁺ (TC) were kept in contact with each other, they fused into a single aggregate in the medium with Ca²⁺ but not without Ca²⁺. Embryos treated with trypsin in the absence of Ca²⁺ (TE) did not show such Ca²⁺-dependent aggregation. Aggregation of TC-treated embryos was inhibited by Fab fragments of antibody raised against TC-treated teratocarcinoma F9 cells. The aggregation-inhibitory effect of the Fab was removed by absorption with TC-treated teratocarcinoma cells, but not with TE-treated teratocarcinoma cells. This effect was not removed by absorption with fibroblasts and some other tissue cells. TC-treated embryos adhered to TC-treated teratocarcinoma cells, but not to TC-treated fibroblastic cells. These results suggest that early mouse embryos share a common CDS molecule with teratocarcinoma cells but not with fibroblastic cells.     

Two distinct adhesion mechanisms in embryonic neural retina cells. I. A kinetic analysis

The reaggregation kinetics of embryonic chick neural retina cells prepared using several different dissociation procedures were monitored through decreases in the small-angle light scattering of aggregating samples. Two distinct modes of aggregation were revealed, one Ca²⁺ independent, the other Ca²⁺ dependent, suggesting the existence of two separate adhesion mechanisms. By varying the concentrations of Ca²⁺ and trypsin in the dissociation medium, we obtained cells which exhibited both, either, or neither mode of aggregation. The Ca²⁺-independent adhesiveness is active in the absence of proteolysis, is resistant to low levels of trypsin (0.001%), but is readily inactivated at higher trypsin concentrations in either the presence or absence of Ca²⁺. It is relatively temperature independent. By contrast, the Ca²⁺-dependent adhesiveness is not detected before exposure of the cells to proteolysis. It is expressed after tryptic proteolysis in the presence of Ca²⁺ and is then highly temperature dependent. It is resistant to further digestion by trypsin in the continued presence of Ca²⁺ but is lost when Ca²⁺ is subsequently removed, apparently through the expression of tryptic cleavage incurred earlier. We suggest that its increased activity may result at least in part from the clustering of surface components into adhesive patches. A provisional model is presented correlating these data.     

Teratocarcinoma cell adhesion: Identification of a cell-surface protein involved in calcium-dependent cell aggregation

Teratocarcinoma cells have a Ca²⁺-dependent cell-cell adhesion site (t-CDS) that is unique in being inactivated with trypsin in the absence of Ca²⁺ but not in the presence of Ca²⁺. Fab fragments of antibodies raised against teratocarcinoma F9 cells dissociated by treatment with trypsin and calcium (anti-TC-F9) inhibit the aggregation of teratocarcinoma cells mediated by t-CDS. This inhibitory effect of Fab is removed when anti-TC-F9 is absorbed with F9 cells treated with trypsin and calcium (TC-F9), but not when it is absorbed with F9 cells treated with trypsin and EGTA (TE-F9). Comparisons of cell-surface antigens reactive to anti-TC-F9 in TC-F9 cells with those in TE-F9 cells reveal that only one component, with an approximate molecular weight of 140,000 (p140), is detected specifically on the surface of TC-F9 cells. When TC-F9 cells are retrypsinized in the absence of Ca²⁺, a substance with an approximate molecular weight of 34,000 (p34) is released that can neutralize the aggregation-inhibitory effect of the Fab. This p34 interferes with the immunoprecipitation of p140 with anti-TC-F9, suggesting that p34 is a tryptic fragment of p140. Anti-TC-F9 Fab causes the dissociation of the monolayers of teratocarcinoma cells. This effect is removed by absorption of the Fab with p34 as well as with TC-F9 cells, but not with TE-F9 cells. These results suggest that p140 is essential for the function of t-CDS, and that this is an actual cell-adhesion molecule active in the establishment of monolayers of teratocarcinoma cells.     

Cell-Cell adhesion molecule: Identification of a glycoprotein relevant to the Ca2+-independent aggregation of chinese hamster fibroblasts

Molecules making up the Ca²⁺-independent cell-cell adhesion sites (CIDS) of Chinese hamster V79 cells have been investigated. Previous studies showed that Fab fragments (Fab) of the antibody raised against the surface of V79 cells inhibit the Ca²⁺-independent aggregation of V79 cells, and that this inhibitory effect is neutralized by the addition of some proteinous substance released from the surface of V79 cells treated with 0.01% trypsin. In the present study, we found that the antibody raised against this substance (anti-TRF) displays the complement-dependent cytotoxicity more strongly to the cells with active CIDS than to those without it. The Fab of this antibody inhibited the Ca²⁺-independent aggregation of V79 cells strongly, particularly in the presence of a hyaluronidase. Electrophoresis of the materials immunoprecipitated with anti-TRF from lysates of cells labeled with radioactive iodine, monosaccharides or methionine showed that a glycoprotein with a molecular weight of 125,000 is the only component to react with this antibody in cells with the active CIDS. Indirect immunofluorescence of anti-TRF showed that the antigens are distributed over the entire surface of V79 cells, and are present only in fibroblastic cells in the primary culture of Chinese hamster hepatic cells. These results suggest that the glycoprotein with a molecular weight of 125,000 has a specific role in the Ca²⁺-independent adhesion of Chinese hamster fibroblastic cells.     

In vitro reaggregation of dissociated mouse cerebellar cells : I. Demonstration of different aggregation mechanisms

Reaggregation of mechanically dissociated mouse cerebellar cells (M cells) was compared with cells that received an additional trypsinization either before (T cells) or after (MT cells) the dissociation step. Reaggregation behaviour was followed by measuring the number and size distribution of particles with a Coulter counter. Aggregation rates which were calculated as percentage of decrease of particles could be measured reproducibly. Since the percentage of very large particles (> 100 cells) formed during aggregation varied considerably from one experiment to the next, size distribution curves of particles were used more to distinguish qualitative differences in a less quantitative way.Whereas aggregation rates and size distribution of particles with M cells were almost identical when aggregation occurred in medium of high (1.1 mM) or low (0.1 mM) Ca²⁺ concentrations, T and MT cells aggregated better at high Ca²⁺ concentration. Their aggregation rates were reduced by approx. 50% at low Ca²⁺ concentrations and larger aggregates were hardly formed under these conditions. The aggregation rates of T and MT cells showed a clear dependence on Ca²⁺ concentration, being half maximal at approx. 0.1 mM Ca²⁺.The ability of M cells to aggregate at low or high Ca²⁺ concentrations was influenced by subsequent trypsinization to produce MT cells. When the trypsin concentration was changed from 0.001 to 0.1% during this procedure the aggregation rates at high Ca²⁺ concentration were reduced to approx. 80% of the maximal value, whereas those at low Ca²⁺ concentrations were reduced to 35%. Variation of the Ca²⁺ concentration between 1.1 and 0.1 mM during the trypsinization step (0.015% trypsin) revealed no difference on the aggregation rates.We propose that M cells aggregate mainly or exclusively by a Ca²⁺-independent binding mechanism, whereas T or MT cells aggregate using a Ca²⁺-dependent one which may be functionally silent in M cells.     

Calcium-dependent cyclic nucleotide phosphodiesterase inhibition of basal activity by heat-stable factors from rat cerebrum

The boiled supernatant fraction from rat cerebrum contained factors which inhibited the basal activity of a Ca²⁺-dependent phosphodiesterase from rat cerebrum. Two inhibitory fractions were isolated by DEAE-cellulose or Sephadex chromatography and were deemed proteins, based on their sensitivity to trypsin digestion. The inhibitory fractions eluted from DEAE-cellulose columns prior to the Ca²⁺-dependent activator protein. The inhibitory factors, unlike the activator protein, were stable to heat treatment under alkaline conditions. The inhibitory factors caused both an increase in K_m for cyclic GMP and a decrease in $\text{V}$. In the presence of calcium ions and purified activator protein, the Ca²⁺-dependent phosphodiesterase was not inhibited by the factors, but instead was slightly stimulated. The inhibitory factors caused a slight apparent stimulation of a Ca²⁺-independent phosphodiesterase from rat cerebrum but this proved instead to be a nonspecific stabilizing effect which was mimicked by bovine serum albumin. After prolonged alkaline treatment, the purified activator protein caused a modest Ca²⁺-independent activation of Ca²⁺-dependent phosphodiesterase. The inhibitory factors antagonized the activation of Ca²⁺-dependent phosphodiesterase by alkaline treated activator protein or by lysophosphatidylcholine. The inhibitory factors had no effect on activity of trypsinized Ca²⁺-dependent phosphodiesterase. Of various other proteins, only casein mimicked the effects of the inhibitory factors on phosphodiesterase activity.     

Liposome Aggregation: Promoted by Trypsin and Papain in the Absence of Cations

Abstract

Phospholipid vesicle aggregation is usually mediated by phospholipid-binding proteins such as the annexins in a Ca²⁺-dependent manner. Here, we describe aggregation of unilamellar liposomes by trypsin and papain in the absence of cations. Cations including Ca²⁺ inhibited the aggregation. While both trypsin and papain promoted aggregation of liposomes made of phosphatidylcholine and phosphatidylglycerol, only papain elicited aggregation of liposomes made of exclusively phosphatidylcholine. Incubation of trypsin for 30 min at 37°C destroyed its liposome aggregating activity, similar treatment had no effect on papain's. Chymotrypsin and pepsin had no liposome aggregating activity.     

Effect of extracellular Ca2+ on the morphogenesis of Dictyostelium discoideum.

Effect of extracellular Ca²⁺ on the morphogenesis of the cellular slime mold Dictyostelium discoideum was examined on agar plate. The concentration of Ca²⁺ in agar plate was controlled by keeping the concentration of a chelating reagent EGTA constant and varying the concentration of total calcium. From experiments in which EGTA concentration was kept at 2.0 × 10^?3 M, it was found that by decreasing Ca²⁺ concentration the morphogenesis was modified so that development of the aggregating amebae into fruiting bodies was accelerated and the period of migrating slugs was shortened. Below 1.0 × 10^?3 M of Ca²⁺ concentration, the total number of aggregates initially increased with decreasing Ca²⁺ concentration, reached a maximum at about 3.0 × 10^?7 M of Ca²⁺ concentration and hereafter decreased with decreasing Ca²⁺ concentration. The number of mature fruiting bodies obtained at 36 h period after starvation depends on Ca²⁺ concentration and the total number of aggregates. The cell aggregation initiated at the same time period after starvation even at an extreme case of 1.0 × 10^?8 M of Ca²⁺ concentration as under enough Ca²⁺ supply, while the formation of mature fruiting body was seriously inhibited. These observation suggested that the cAMP-mediated cell aggregation in D. discoideum is a Ca²⁺-independent phenomena, although extracellular Ca²⁺ is necessary for the normal development of the aggregated amebae.     

Experimental manipulation of cell surface to affect cellular recognition mechanisms.

The mechanism of selective cell adhesion was studied using Chinese hamster V79 and chick embryonic neural retinal cells. Both of these cell types have been shown to have two experimentally separable mechanisms of adhesion; Ca²⁺-dependent and Ca²⁺-independent. Cells can be dispersed so that either or both of the mechanisms remain intact by use of different treatments. A method of labeling cells with FITC was devised to identify one of the two types of cells in a binary cell population. When cells with one of the two adhesion mechanisms were mixed with cells with the other mechanism, they segregated completely, forming independent aggregates, not only in the heterotypic combination of these cell types but also in the homotypic combination of each cell type. In contrast, when cells were mixed with others with the same adhesion mechanism, either Ca²⁺-dependent or -independent, they formed chimeric aggregates, even in the heterotypic cell combination. These results suggest that the specificity in each of those two mechanisms of cell adhesion plays an important role in cellular recognition processes.     

CALCIUM-INDUCED RELEASE OF γ-AMINOBUTYRIC ACID FROM SYNAPTOSOMES: EFFECTS OF TRANQUILIZER DRUGS

Efflux of preloaded radioactive gamma-aminobutyric acid (GABA) from mouse brain fractions enriched in synaptosomes could be significantly and reproducibly increased by switching from Ca²⁺-free wash solution to one containing 2 mM-Ca²⁺. With a filtration apparatus equipped for simultaneous buffer changes of eight samples, effects of various drugs on Ca²⁺-dependent and Ca²⁺-independent efflux of GABA were studied. Several membrane-perturbing agents, including imipramine and neuroleptics like haloperidol and chlorpromazine, at 1 μM, potently inhibited Ca²⁺-dependent GABA release. Benzodiazepine drugs similarly inhibited Ca²⁺-dependent efflux, but also under appropriate conditions, enhanced Ca²⁺-independent efflux. Stimulation of Ca²⁺-independent efflux was observed with nonradioactive GABA and with GABA analogues which inhibit GABA transport, reflecting exchange of GABA via the high-affinity transport system. Diazepam was found to inhibit GABA transport (non-competitively; I₅₀, 50 μM); effects on the transport system might be related to observations of potentiated GABA action and anti-convulsant effects of this drug.     

Purification and characterization of a species-specific aggregation factor in sponges

The events during re-aggregation of sponge cells, dissociated in Ca²⁺- and Mg²⁺-free artificial sea water, containing trypsin, can be subdivided into three phases. The first event is the formation of primary aggregates with a diameter of 82 μm. It is Ca²⁺- and Mg²⁺-dependent and insensitive towards trypsin and puromycin even at high concentrations. The formation of secondary aggregates with a diameter greater than 1 000 μm, is initiated by an aggregation factor. This factor can be separated from the formed elements with calcium- and magnesium-free artificial sea water. Its action is Ca²⁺- and Mg²⁺-dependent, temperature-independent and insensitive to puromycin. The last event is the reconstitution of functional aquiferous systems in the aggregates.The aggregation factor which was found to be species-specific could be purified 158-fold. Its functional group seems to be a protein probably with polar amino acids in critical positions. Secondary aggregates generated with the aggregation factor show high viability. Some evidence is presented that the aggregation factor may be an annular particle with a circular contour length of 3 500 Å with about 25 filaments attached to it.     

Calcium-dependent cell-cell adhesion molecules common to hepatocytes and teratocarcinoma stem cells

The molecules involved in Ca2+-dependent cell-cell adhesion systems (CDS) in mouse hepatocytes were characterized and compared with those in teratocarcinoma cells. Fab fragments of antibody raised against liver tissues (anti-liver) inhibited Ca2+-dependent aggregation of both liver and teratocarcinoma cells. A monoclonal antibody raised against teratocarcinoma CDS (ECCD-1) also inhibited the Ca2+-dependent aggregation of these two cell types equally. These antibodies induced disruption of cell-cell adhesion in monolayers of hepatocytes. Thus, CDS in these two cell types are not immunologically distinctive. Immunochemical analyses with these antibodies showed that CDS in both hepatocytes and teratocarcinoma cells involved at least two classes of cell surface proteins with molecular weights of 124,000 and 104,000. ECCD-1 selectively bound to hepatocytes but not to fibroblastic cells in liver cell cultures. Thus, the molecular constitution of CDS in hepatocytes and teratocarcinoma stem cells is identical. As ECCD-1 reacts with other classes of embryonic and fetal cells, the molecules identified here could have a major role in cell-cell adhesion in various tissues at any developmental stage of animals.     

Identification of platelet membrane thrombospondin binding molecules using an anti-thrombospondin antibody

A rat monoclonal IgG_2a antibody, 5G11, was raised against native human platelet thrombospondin (TSP). Western blot analysis revealed that 5G11 bound (i) to TSP before and after disulfide reduction, and (ii) to a 15-kDa fragment released after prolonged trypsin digestion. Crossed immunoelectrophoresis confirmed that the binding epitope was expressed in the presence of Ca²⁺ and after treatment of TSP with EDTA. Since 5G11 had no effect on platelet aggregation, the antibody was used to immunoprecipitate Ca²⁺-dependent and Ca²⁺-independent TSP-binding molecules on the surface of thrombin-activated surface-labeled ¹²⁵I-platelets. The experimental basis was that ligand-receptor interactions are of high affinity and that anti-ligand antibodies should precipitate the ligand-receptor complex. With platelets activated in the presence of EDTA, 5G11 predominantly precipitated a ¹²⁵I-labeled band of M_r 88 000, identified as glycoprotein (GP) IV. In contrast, in the presence of 2 mM Ca²⁺ and 1 mM Mg²⁺, 5G11 precipitated a complex of five radiolabeled proteins, among which GPIIb, GPIIIa and GPIV were the most prominent.     

Requirements for the Ca2+-independent component in the initial intercellular adhesion of C2 myoblasts

Using a sensitive and quantitative adhesion assay, we have studied the initial stages of the intercellular adhesion of the C2 mouse myoblast line. After dissociation in low levels of trypsin in EDTA, C2 cells can rapidly reaggregate by Ca2+-independent mechanisms to form large multicellular aggregates. If cells are allowed to recover from dissociation by incubation in defined media, this adhesive system is augmented by a Ca2+-dependent mechanism with maximum recovery seen after 4 h incubation. The Ca2+-independent adhesion system is inhibited by preincubation of cell monolayers with cycloheximide before dissociation. Aggregation is also reduced after exposure to monensin, implicating a role for surface-translocated glycoproteins in this mechanism of adhesion. In coaggregation experiments using C2 myoblasts and 3T3 fibroblasts in which the Ca2+-dependent adhesion system was inactivated, no adhesive specificity between the two cell types was seen. Although synthetic peptides containing the RGD sequence are known to inhibit cell-substratum adhesion in various cell types, incubation of C2 myoblasts with the integrin-binding tetrapeptide, RGDS, greatly stimulated the Ca2+-independent aggregation of these cells while control analogs had no effect. These results show that a Ca2+- independent mechanism alone is sufficient to allow for the rapid formation of multicellular aggregates in a mouse myoblast line, and that many of the requirements and perturbants of the Ca2+-independent system of intercellular myoblast adhesion are similar to those of the Ca2+-dependent adhesion mechanisms.     

Effects of staurosporine on Ca2+-dependent and Ca2+-independent [14C]GABA release from rat brain synaptosomes

The effect of a protein kinase inhibitor, staurosporine, on Ca²⁺-dependent and Ca²⁺-independent release of [¹⁴C]GABA in isolated rat brain synaptosomes was studied. Calcium-dependent [¹⁴C]GABA release was stimulated by depolarization with a K⁺ channel blocker, 4-aminopyridine (4-AP), or high K⁺ concentration. It has been shown that the effect of 4-AP is Ca²⁺-dependent, while high K⁺ is able to evoke [¹⁴C]GABA release in both Ca²⁺-dependent and Ca²⁺-independent manners. In addition, Ca²⁺-independent [¹⁴C]GABA release was studied using α-latrotoxin (LTX) as a tool. Pretreatment of synaptosomes with staurosporine resulted in pronounced inhibition of 4-AP-stimulated Ca²⁺-dependent [¹⁴C]GABA release. The inhibitory effect of staurosporine on [¹⁴C]GABA release was not due to modulation of 4-AP-promoted⁴⁵Ca²⁺ influx into synaptosomes. If the process of [¹⁴C]GABA release occurred in the Ca²⁺-independent manner irrespectively of what, LTX or high K⁺, stimulated this process, it was not inhibited by staurosporine. Considering the above findings, it is reasonable to assume that the absence of Ca²⁺ in the extracellular medium created conditions for activation of the process of neurotransmitter release without Ca²⁺-dependent dephosphorylation of neuronal phosphoproteins; as a consequence, regulation of exocytotic process was modulated in such a manner that inhibition of protein kinases did not disturb exocytosis.     

Calcium-dependent adhesion of Drosophila embryonic cells

Summary By using an in vitro functional assay, we have shown that Drosophila embryonic cells possess Ca²⁺-dependent adhesive sites, which resemble in many respects those described for vertebrate cells and tissues. The cells, obtained by mechanical disruption of gastrulastage embryos, form aggregates within 30 min when maintained under constant rolling. The aggregation is completely dependent on the presence of Ca²⁺ in the medium. In its absence, the cells remain dispersed but the process is reversible by readdition of Ca²⁺. In addition the aggregation is temperature-dependent. No aggregation occurs at 4° C but it can be restored by raising the temperature to 25° C. These properties are characteristic of these cells: established cell lines do not aggregate under the same conditions and mixing of cell lines and embryonic cells does not result in chimeric aggregates, thus pointing towards cell-type selectivity with respect to aggregability. Observations in electron microscopy have shown that the embryonic cells in the aggregates tightly adhere to one another and form, as early as after 30 min, maculae adherens junctions. Drosophila embryonic cells have adhesion sites that are protected from trypsin proteolysis in the presence of Ca²⁺ and sensitive in its absence. The cells' aggregation can be inhibited by a mouse antiserum directed against cell-surface components and a good correlation exists between neutralization of the inhibitory activity of the antiserum and the presence of trypsin-sensitive sites on the cells. These data are in favour of cell-cell adhesion mediated by specific adhesion proteins.     

A comparative study of the rat heart sarcolemmal Ca2+-dependent ATPase and myosin ATPase

In order to gain some information regarding Ca²⁺-dependent ATPase, the enzyme was purified from cardiac sarcolemma and its properties were compared with Ca²⁺-ATPase activity of myosin purified from rat heart. Both Ca²⁺-dependent ATPase and myosin ATPase were stimulated by Ca²⁺ but the maximal activation of Ca²⁺-dependent ATPase required 4 mM Ca²⁺ whereas that of myosin ATPase required 10 mM Ca²⁺. These ATPases were also activated by other divalent cations in the order of Ca²⁺ > Mn²⁺ > Sr²⁺ > Br²⁺ > Mg²⁺; however, there was a marked difference in the pattern of their activation by these cations. Unlike the myosin ATPase, the ATP hydrolysis by Ca²⁺-dependent ATPase was not activated by actin. The pH optima of Ca²⁺-dependent ATPase and myosin ATPase were 9.5 and 6.5 respectively. Na⁺ markedly inhibited Ca²⁺-dependent ATPase but had no effect on the myosin ATPase activity. N-ethylmaleimide inhibited Ca²⁺-dependent ATPase more than myosin ATPase whereas the inhibitory effect of vanadate was more on myosin ATPase than Ca²⁺-dependent ATPase. Both Ca²⁺-dependent ATPase and myosin ATPase were stimulated by K-EDTA and NH₄-EDTA. When myofibrils were treated with trypsin and passed through columns similar to those used for purifying Ca²⁺-ATPase from sarcolemma, an enzyme with ATPase activity was obtained. This myofibrillar ATPase was maximally activated at 3–4 mM Ca²⁺ and 3 to 4 mM ATP like sarcolemmal Ca²⁺-dependent ATPase. K⁺ stimulated both ATPase activities in the absence of Ca²⁺ and inhibited in the presence of Ca²⁺. Both enzymes were inhibited by Na⁺, Mg²⁺, La³⁺, and azide similarly. However, Ca²⁺ ATPase from myofibrils showed three peptide bands in SDS polyacrylamide gel electrophoresis whereas Ca²⁺ ATPase from sarcolemma contained only two bands. Sarcolemmal Ca²⁺-ATPase had two affinity sites for ATP (0.012 mM and 0.23 mM) while myofibrillar Ca²⁺-ATPase had only one affinity site (0.34 mM). Myofibrillar Ca²⁺-ATPase was more sensitive to maleic anhydride and iodoacetamide than sarcolemmal Ca²⁺-ATPase. These observations suggest that Ca²⁺-dependent ATPase may be a myosin like protein in the heart sarcolemma and is unlikely to be a tryptic fragment of myosin present in the myofibrils.     

Magnesium-dependent induction of phagocytosis-associated chemiluminescence of adherent human polymorphonuclear leukocytes by non-opsonized zymosan

A severe dysfunction in the cellular response of human polymorphonuclear leukocytes (PMNL) to non-opsonized zymosan was observed under a deficiency of extracellular Mg²⁺. The phagocytosis-association native (luminol-independent) luminescence (NL), as well as luminol-dependent luminescence (LDL) (detected simultaneously and discriminated by spectral methods), was strongely inhibited. Apart from a general decrease of total light production, a Mg²⁺-concentration-dependent delay of the maximum of NL and LDL was observed. A disorder in recruitment of activated membrane-bound NADPH-oxidase of PMNL is suggested. The presence of extracellular Ca²⁺ did not compensate for the Mg²⁺ deficit. In the presence of Mg²⁺ only a slight Ca²⁺-dependent reduction of NL was obtained, but Ca²⁺ seemed to selectively promote LDL. This may indicate a positive influence of Ca²⁺ on the myeloperoxidase release from the cells. Experiments with the metalions-chelating agents EDTA and EGTA, which complex Mg²⁺ to differing extents, confirmed the important role of Mg²⁺ in PMNL-activation by non-opsonized zymosan.     

Effects of low temperature and calcium on microfilament structure in flagellates of Physarum polycephalum

Microfilament structures of the ridge and the backbone in Physarum flagellates disintegrated selectively within a few minutes upon cooling by ice-water. The elongated cells concurrently rounded up to spherical or irregular and amoeboid shape. When such rounded cells were warmed to 25 °C, the microfilament structures were reconstructed within 1 min and cells subsequently returned to an elongated shape. Disruption of microfilaments by cytochalasin A also caused the rounding up of cells, indicating that the rounding up resulted from disintegration of microfilament structures. This transformation induced by the cold treatment was retarded by preincubation of the cells with EGTA for 15 min, but addition of EGTA immediately before the onset of the cold treatment was less effective. The effect of EGTA was cancelled by simultaneous addition of excess Ca²⁺. Addition of procaine also inhibited the transformation induced by the cold treatment, while caffeine inhibited the recovery of the elongated shape when returned to 25 °C. Furthermore, addition of A23187 at 25 °C in the presence of Ca²⁺ mimicked the effect of the cold treatment. Thus, intracellular release of Ca²⁺ was suggested to be involved in the transformation induced by the cold treatment. Lability of the microfilament structures at a high concentration of Ca²⁺ was directly proved using Triton-permeabilized cells. Therefore we concluded that low temperature disrupts microfilament structures that are necessary for the maintenance of the elongated cell shape by inducing intracellular Ca²⁺ release. However, microfilament structures in Physarum amoeba cells were affected neither by the cold treatment nor by high Ca²⁺ concentration.