Ca2+ signaling induced by sphingosine 1-phosphate and lysophosphatidic acid in mouse B cells

Lysophospholipids (LPLs) such as lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) are chemotactic for lymphocytes, and increases of in cytosolic [Ca²⁺] signal the regulation of lymphocyte activation and migration. Here, the authors investigated the effects of LPA and S1P on [Ca²⁺]_c in mouse B cell lines (WEHI-231 and Bal-17) and primary B cells isolated from mouse spleen and bone marrow, and focused on the modulation of store-operated Ca²⁺ entry (SOCE) by LPLs. In Bal-17 (a mature B cell line) both LPA and S1P induced a transient [Ca²⁺]_c increase via a phospholipase C pathway. In addition, pretreatment with LPLs was found to augment thapsigargin-induced SOCE in Bal-17 cells. However, in WEHI-231 (an immature B cell line) LPLs had no significant effect on [Ca²⁺]_c or SOCE. Furthermore, in freshly isolated splenic B cells (SBCs) and bone marrow B cells (BMBCs), LPLs induced only a small increase in [Ca²⁺]_c. Interestingly, however, pretreatment with LPLs markedly increased SOCE in primary B cells, and this augmentation was more prominent in BMBCs than SBCs. The unidirectional influx of Ca²⁺ was measured using Ba²⁺ as a surrogate ion. Similarly, Ba²⁺ influx was also found to be markedly increased by LPLs in SBCs and BMBCs. Summarizing, LPLs were found to strongly augment SOCE-mediated Ca²⁺-signaling in mouse B cells. However, unlike the mature Bal-17 cell line, PLC-dependent Ca²⁺ release was insignificant in primary B cells and inWEHI-231.     

Oligosaccharides related to xyloglucan: synthesis and X-ray crystal structure of methyl alpha-L-fucopyranosyl-(1-->2)-beta-D-galactopyranosyl-(1-->2)-alpha-D-x ylopyranoside and the synthesis of methyl alpha-L-fucopyranosyl-(1-->2)-beta-D-galactopyranosyl-(1-->2)-beta-D-xy lopyranoside

Trisaccharides, methyl alpha-L-fucopyranosyl-(1-->2)-beta-D-galactopyranosyl-(1-->2)-alpha-D-xy lopyranoside and methyl alpha-L-fucopyranosyl-(1-->2)-beta-D-galactopyranosyl-(1-->2)-beta-D-xyl opyranoside, which are related to the side chain of xyloglucan have been synthesised. The beta-galactopyranosyl linkage of each was constructed using silver trifluoromethanesulfonate-promoted glycosylations of 2-O-acetyl-3,4,6-tri-O-benzyl-beta-D-galactopyranosyl chloride and the corresponding anomer of methyl 3,4-tri-O-benzyl-D-xylopyranoside. The resulting disaccharides were deacetylated and fucosylated using assisted halide reactions with tri-O-benzyl-alpha-L-fucopyranosyl bromide. Hydrogenolytic debenzylation of the resulting protected trisaccharides gave the methyl glycosides of the fucose-containing xyloglucan side chain. The structure of methyl alpha-L-fucopyranosyl-(1-->2)-beta-D-galactopyranosyl-(1-->2)-alpha-D-xy lopyranoside as the monohydrate was confirmed by an X-ray crystallographic study.     

Antioxidant dieckol downregulates the Rac1/ROS signaling pathway and inhibits Wiskott-Aldrich syndrome protein (WASP)-family verprolin-homologous protein 2 (WAVE2)-mediated invasive migration of B16 mouse melanoma cells

Reactive oxygen species (ROS) generation is linked to dynamic actin cytoskeleton reorganization, which is involved in tumor cell motility and metastasis. Thus, inhibition of ROS generation and actin polymerization in tumor cells may represent an effective anticancer strategy. However, the molecular basis of this signaling pathway is currently unknown. Here, we show that the Ecklonia cava-derived antioxidant dieckol downregulates the Rac1/ROS signaling pathway and inhibits Wiskott-Aldrich syndrome protein (WASP)-family verprolin-homologous protein 2 (WAVE2)-mediated invasive migration of B16 mouse melanoma cells. Steady-state intracellular ROS levels were higher in malignant B16F10 cells than in parental, nonmetastatic B16F0 cells. Elevation of ROS by H₂O₂ treatment increased migration and invasion ability of B16F0 cells to level similar to that of B16F10 cells, suggesting that intracellular ROS signaling mediates the prometastatic properties of B16 mouse melanoma cells. ROS levels and the cell migration and invasion ability of B16 melanoma cells correlated with Rac1 activation and WAVE2 expression. Overexpression of dominant negative Rac1 and depletion of WAVE2 by siRNA suppressed H₂O₂-induced cell invasion of B16F0 and B16F10 cells. Similarly, dieckol attenuates the ROS-mediated Rac1 activation and WAVE2 expression, resulting in decreased migration and invasion of B16 melanoma cells. In addition, we found that dieckol decreases association between WAVE2 and NADPH oxidase subunit p47^phox. Therefore, this finding suggests that WAVE2 acts to couple intracellular Rac1/ROS signaling to the invasive migration of B16 melanoma cells, which is inhibited by dieckol.     

Effect of amphotericin B on dopachrome tautomerase activity and other melanogenic parameters in cultured B16/F10 melanoma cells.

The antifungal reagent Fungizone (amphotericin B and deoxycholate) caused an activation in dopachrome tautomerase and dopa oxidase activities of B16/F10 melanoma cells at the routine concentration (2.5 micrograms/ml) used for preventing molds and yeast growth in cultures of animal cells. However, higher amphotericin B concentrations caused a significant cell death and the inhibition of enzymatic activities. At the optimal concentration of Fungizone, the enzymatic activities and melanin content were augmented as incubation time increased. The detergent sodium deoxycholate alone exerted no effect on these melanogenic parameters, eliminating the possibility that this detergent was partially responsible for melanogenic modifications produced by Fungizone. After withdrawal of Fungizone from the reaction medium, the recovery of melanogenic parameters to normal values was slower for DCT than for tyrosinase. The behavior of dopa oxidase was very similar to that reported by Johnson and Bagnara (Pigment Cell Res. 3, 173-175) for tyrosine hydroxylase.     

Tyrosinase-related protein-2 and -1 are trafficked on distinct routes in B16 melanoma cells

Tyrosinase related protein (TRP)-1 and -2 regulate the main steps in melanin synthesis and are immune targets in skin cancer or autoimmune pigmentary disorders. We found that ionophore monensin (Mon) and the quaternary amine chloroquine (CQ) discriminate between the traffic routes of TRP-2 and TRP-1. TRP-2 N-glycan processing is interrupted by Mon between ER and trans-Golgi, whereas this process continues for TRP-1. Mature TRP-2 is diverted by CQ treatment to a degradation pathway which depends on functional vacuolar ATPases. Conversely, the subcellular distribution and stability of TRP-1 were not affected by CQ. We propose that TRP-2 is sorted and trafficked in the early secretory pathway with a cargo which does not include TRP-1; post Golgi, TRP-2 intersects the endocytic pathway following a route via early endosomes, possibly by rapid recycling from the plasma membrane. These data show that highly structural homologous glycoproteins use distinct trafficking pathways in the same cell.     

Synthesis of alpha-Manp-(1-->2)-alpha-Manp-(1-->3)-alpha-Manp-(1-->3)-Manp, the tetrasaccharide repeating unit of Escherichia coli O9a, and alpha-Manp-(1-->2)-alpha-Manp-(1-->2)-alpha-Manp-(1-->3)-alpha-Manp-(1-->3)-Manp, the pentasaccharide repeating unit of E. coli O9 and Klebsiella O3

The tetrasaccharide repeating unit of Escherichia coli O9a, alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->3)-alpha-D-Manp-(1-->3)-D-Manp, and the pentasaccharide repeating unit of E. coli O9 and Klebsiella O3, alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->3)-alpha-D-Manp-(1-->3)-D-Manp, were synthesized as their methyl glycosides. Thus, selective 3-O-allylation of p-methoxyphenyl alpha-D-mannopyranoside via a dibutyltin intermediate gave p-methoxyphenyl 3-O-allyl-alpha-D-mannopyranoside (2) in good yield. Benzoylation (-->3), then removal of 1-O-methoxyphenyl (right arrow4), and subsequent trichloroacetimidation afforded the 3-O-allyl-2,4,6-tri-O-benzoyl-alpha-D-mannopyranosyl trichloroacetimidate (5). Condensation of 5 with methyl 4,6-O-benzylidene-alpha-D-mannopyranoside (6) selectively afforded the (1-->3)-linked disaccharide 7. Benzoylation of 7, debenzylidenation, benzoylation, and deallylation gave methyl 2,4,6-tri-O-benzoyl-alpha-D-mannopyranosyl-(1-->3)-2,4,6-tri-O-benzoyl-alpha-D-mannopyranoside (11) as the disaccharide acceptor. Coupling of 11 with (1-->2)-linked mannose disaccharide donor 17 or trisaccharide donor 21, followed by deacylation, furnished the target tetrasaccharide and pentasaccharide, respectively.     

Antimelanomal activity of the copper(II) complexes of 1-substituted 5-amino-imidazole ligands against B16F10 mouse melanoma cells

The copper complexes of 5-amino-imidazole ligands were prepared and characterized by various spectroscopic techniques. The ligand geometry around the copper(II) centre is square pyramidal based on N2O2 donor atoms and a coordinated water molecule at the apex. Single crystal X-ray structures were determined for both ligands. Ligands and copper complexes exhibited dose-dependent antiproliferative effects on the growth of B16F10 melanoma cells line but lower IC50 values were observed for the copper complexes.     

Probing the substrate specificity of four different sialyltransferases using synthetic beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->2)-alpha-D-Manp-(1-->O) (CH(2))7CH3 analogues general activating effect of replacing N-acetylglucosamine by N-propionylglucosamine

The acceptor specificities of ST3Gal III, ST3Gal IV, ST6Gal I and ST6Gal II were investigated using a panel of beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->2)-alpha-D-Manp-(1-->O)(CH(2))(7)CH(3) analogues. Modifications introduced at either C2, C3, C4, C5, or C6 of terminal D-Gal, as well as N-propionylation instead of N-acetylation of subterminal D-GlcN were tested for their influence on the alpha-2,3- and alpha-2,6-sialyltransferase acceptor activities. Both ST3Gal enzymes displayed the same narrow acceptor specificity, and only accept reduction of the Gal C2 hydroxyl function. The ST6Gal enzymes, however, do not have the same acceptor specificity. ST6Gal II seems less tolerant towards modifications at Gal C3 and C4 than ST6Gal I, and prefers beta-D-GalpNAc-(1-->4)-beta-D-GlcpNAc (LacdiNAc) as an acceptor substrate, as shown by replacing the Gal C2 hydroxyl group with an N-acetyl function. Finally, a particularly striking feature of all tested sialyltransferases is the activating effect of replacing the N-acetyl function of subterminal GlcNAc by an N-propionyl function.     

Expression of the cell adhesion molecules N-CAM and L1 in B16 melanoma cells

The cell adhesion molecules N-CAM and L1 are important for cell-cell recognition and cell migration and so may be involved in the metastatic process. We have studied the biosynthesis of N-CAM and L1 in the B16 melanoma cell lines B16-F1 and B16-F10 which differ in metastatic capacity. N-CAM was synthesised as two glycosylated polypeptides with Mr of 150,000 and 210,000; L1 was synthesised as one polypeptide with Mr of 215,000. In fetal neurons N-CAM is synthesised as a 135,000 and a 200,000 Mr polypeptide and L1 as a 200,000 Mr polypeptide. Thus, the Mr of N-CAM and L1 in tumour cells appeared to be 10,000-15,000 higher than in the normal cells. L1 was phosphorylated in the tumour cells as in neurons. The tumour cells also phosphorylated the 210,000 Mr N-CAM polypeptide, whereas no phosphorylation of the 150,000 Mr polypeptide was observed. In neuronal cells both the corresponding polypeptides are phosphorylated and thus the biosynthesis of N-CAM in tumour cells seem to differ from that in neuronal cells with regard to phosphorylation. No differences in biosynthesis of N-CAM or L1 were apparent between the two tumour cell lines, B16-F1 and B16-F10.     

Alpha-D-mannopyranosyl-(1-->2)-alpha-D-glucopyranosyl-(1-->2)-glycerate in the thermophilic bacterium Petrotoga miotherma--structure, cellular content and function

The intracellular accumulation of low molecular mass organic compounds in response to stressful conditions was investigated in the thermophilic bacterium Petrotoga miotherma, a member of the order Thermotogales. This led to the discovery of a new solute, whose structure was established as alpha-D-mannopyranosyl-(1-->2)-alpha-D-glucopyranosyl-(1-->2)-glycerate (MGG) by MMR spectroscopy and MS. Under optimum growth conditions (3% NaCl; 55 degrees C), MGG was the major solute [up to 0.6 micromol.(mg protein)(-1)]; alpha-glutamate and proline were also present but in minor amounts [below 0.08 micromol.(mg protein)(-1)]. The level of MGG increased notably with the salinity of the growth medium up to the optimum NaCl concentration. At higher NaCl concentrations, however, the level of MGG decreased, whereas the levels of proline and alpha-glutamate increased about five-fold and 10-fold, respectively. MGG plays a role during low-level osmotic adaptation of Petrotoga miotherma, whereas alpha-glutamate and, to a lesser extent, proline are used for osmoprotection under salt stress. MGG is not part of the cell strategy for coping with heat or oxidative stress. Nevertheless, MGG was an efficient protector of pig heart malate dehydrogenase against heat inactivation and freeze-drying, although mannosylglycerate was better. This is the first report on the occurrence of MGG in living systems.     

Alteronol inhibits the invasion and metastasis of B16F10 and B16F1 melanoma cells in vitro and in vivo

Aims

The purpose of this study is to evaluate the anti-metastatic effects of alteronol on melanoma B16F10 and B16F1 cells in vitro and in vivo.

Main methods

Melanoma B16F1 and B16F10 cells were cultured in vitro. Cell proliferation was analyzed via 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. The cell migration and invasion were evaluated via wound healing and transwell chamber assays. The activity of matrix metalloproteinase 2 (MMP-2) in culture supernatants was assessed via gelatin zymography. The expression of MMP-2 and TIMP-2 were detected via enzyme-linked immunosorbent assay (ELISA) assay. The anti-metastatic ability in vivo was detected through experimental lung metastasis.

Key findings

The data indicate that alteronol can inhibit the proliferation, invasion, and migration of B16F1 and B16F10 cells in vitro and in vivo, decrease the activity and expression of MMP-2, enhance the expression level of Tissue Inhibitor of Metalloproteinase-2 (TIMP-2), and inhibit the experimental lung metastasis of B16F1 and B16F10 cells.

Significance

Although alteronol and taxol are obtained from the same source, these substances do not destroy the rare resource; the mechanisms of them on tumor growth inhibition are different. Conversely, alteronol treatment had lesser effects on normal cells revealing for a selective property and a strong competitive advantage.     

Down-regulation of glutathione and Bcl-2 synthesis in mouse B16 melanoma cells avoids their survival during interaction with the vascular endothelium

B16 melanoma (B16M) cells with high GSH content show high metastatic activity. However, the molecular mechanisms linking GSH to metastatic cell survival are unclear. The possible relationship between GSH and the ability of Bcl-2 to prevent cell death was studied in B16M cells with high (F10) and low (F1) metastatic potential. Analysis of a Bcl-2 family of genes revealed that B16M-F10 cells, as compared with B16M-F1 cells, overexpressed preferentially Bcl-2 (approximately 5.7-fold). Hepatic sinusoidal endothelium-induced B16M-F10 cytotoxicity in vitro increased from approximately 19% (controls) to approximately 97% in GSH-depleted B16M-F10 cells treated with an antisense Bcl-2 oligodeoxynucleotide (Bcl-2-AS). l-Buthionine (S,R)-sulfoximine-induced GSH depletion or Bcl-2-AS decreased the metastatic growth of B16M-F10 cells in the liver. However, the combination of l-buthionine (S,R)-sulfoximine and Bcl-2-AS abolished metastatic invasion. Bcl-2-overexpressing B16M-F1/Tet-Bcl-2 and B16M-F10/Tet-Bcl-2 cells, as compared with controls, showed an increase in GSH content, no change in the rate of GSH synthesis, and a decrease in GSH efflux. Thus, Bcl-2 overexpression may increase metastatic cell resistance against oxidative/nitrosative stress by inhibiting release of GSH. In addition, Bcl-2 availability regulates the mitochondrial GSH (mtGSH)-dependent opening of the permeability transition pore complex. Death in B16M-F10 cells was sharply activated at mtGSH levels below 30% of controls values. However, this critical threshold increased to approximately 60% of control values in Bcl-2-AS-treated B16M-F10 cells. GSH ester-induced replenishment of mtGSH levels (even under conditions of cytosolic GSH depletion) prevented cell death. Our results indicate that survival of B16M cells with high metastatic potential can be challenged by inhibiting their GSH and Bcl-2 synthesis.     

Transforming growth factor beta1 mediates hypopigmentation of B16 mouse melanoma cells by inhibition of melanin formation and melanosome maturation

Transforming growth factor-beta1 (TGFbeta1) downregulates tyrosinase in B16 melanoma cells by decreasing gene expression and the intracellular half-life of the enzyme, but does not block tyrosinase stimulation by alpha-melanocyte stimulating hormone (alphaMSH). In the presence of both agents, the enzymatic activity is intermediate between the one of cells treated with either agent alone. Here we show that TGFbeta1 equally inhibits the melanogenic activities of melan-a melanocytes and B16 melanoma cells, thus validating the B16 model. In both cell types, TGFbeta1 (10(-10) M, 48 h) inhibited to comparable levels tyrosine hydroxylation and melanin formation from L-tyrosine. Thus, the inhibitory effect is exerted mainly at the rate limiting step of the pathway. By means of quantitative image analysis techniques, we also studied the effects of TGFbeta1 and alphaMSH on melanosome number, volume density and maturation degree. alphaMSH (10(-7) M, 48 h) increased 7-fold melanosome volume density, whereas TGFbeta1 by itself had no significant effect. However, melanosomal volume density was intermediate in cells treated with both agents, as compared to control or alphaMSH-treated cells. Moreover, TGFbeta1 blocked the alphaMSH-elicited increase in the number of melanosomes. Control and alphaMSH-treated melanocytes contained more stage I+II premelanosomes and stage IV, fully melanized organelles than partially melanized stage III melanosomes. TGFbeta1 increased the percentage of stage III melanosomes. This trend was even more marked in cells treated with alphaMSH and TGFbeta1. The accumulation of incompletely melanized melanosomes is consistent with the inhibition of melanin formation activity by TGFbeta1 and with its hypopigmenting effect.     

PRL-3 siRNA inhibits the metastasis of B16-BL6 mouse melanoma cells in vitro and in vivo

Phosphatase of regenerating liver-3 (PRL-3) has been proposed to promote the invasion of tumor cells to metastasis sites. However, the effect of PRL-3 on spontaneous metastasis has not been clearly demonstrated, and whether PRL-3 could become a new therapeutic target in malignant tumor is still unknown. In this study, we used PRL-3 siRNA as a molecular medicine to specifically reduce the expression of PRL-3 in B16-BL6 cells, a highly metastatic melanoma cell line. In vitro, PRL-3 siRNA significantly inhibited cell adhesion and migration, but had no effect on cell proliferation. In the spontaneous metastatic tumor model in vivo, PRL-3 siRNA treatment remarkably inhibited the proliferation of primary tumor, prevented tumor cells from invading the draining lymph nodes, and prolonged the life span of mice. Therefore, our results indicate that PRL-3 plays a critical role in promoting the whole process of spontaneous metastasis and tumor growth initiation, and that inhibiting PRL-3 will improve malignant tumor therapy.     

Primary structure of the mouse laminin B2 chain and comparison with laminin B1

One of the major components of basement membranes is the glycoprotein laminin, made up of three disulfide-bonded subunits, the A, B1, and B2 chains. We have isolated and sequenced overlapping mouse laminin B2 chain cDNA clones covering 7562 base pairs. The deduced amino acid sequence predicts that the mature B2 chain consists of 1572 residues, has an unglycosylated molecular weight of 173,541, and possesses 14 potential N-linked glycosylation sites. Analysis of the predicted secondary structure shows the presence of six domains, two rich in alpha-helical structure, two composed of homologous cysteine-rich repeat units, and two globular regions. The organization of the molecule is very similar to that of the mouse laminin B1 chain, and significant sequence homology between the B1 and B2 chains was found in their two cysteine-rich domains and in their amino-terminal globular domains.     

1.67-A X-ray structure of the B2 immunoglobulin-binding domain of streptococcal protein G and comparison to the NMR structure of the B1 domain.

The structure of the B2 immunoglobulin-binding domain of streptococcal protein G has been determined at 1.67-A resolution using a combination of single isomorphous replacement (SIR) phasing and manual fitting of the coordinates of the NMR structure of B1 domain of streptococcal protein G [Gronenborn, A. M., et al. (1991) Science 253, 657-661]. The final R value was 0.191 for data between 8.0 and 1.67 A. The structure described here has 13 residues preceding the 57-residue Ig-binding domain and 13 additional residues following it, for a total of 83 residues. The 57-residue binding domain is well-determined in the structure, having an average B factor of 18.0. Only residues 8-77 could be located in the electron density maps, with the ends of the structure fading into disorder. Like the B1 domain, the B2 domain consists of four beta-strands and a single helix lying diagonally across the beta-sheet, with a -1, +3 chi, -1 topology. This small structure is extensively hydrogen-bonded and has a relatively large hydrophobic core. These structural observations may account for the exceptional stability of protein G. A comparison of the B2 domain X-ray structure and the B1 domain NMR structure showed minor differences in the turn between strands and two and a slight displacement of the helix relative to the sheet. Hydrogen bonds between crystallographically related molecules account for most of these differences.     

A 2H NMR study on alteration of the membrane organization of mouse B16 melanoma cells treated with 12-O-tetradecanoylphorbol-13-acetate

In order to monitor the membrane fluidity of cells without perturbation by an introduced probe, we developed a method for large-scale preparation of 2H-labeled melanoma cells for a 2H NMR study by incubating melanoma cells with [18,18,18-2H3]stearic acid/phosphatidylcholine liposomes for 2 h at 37 degrees C. It turned out that this treatment did not significantly change the cell viability, lipid metabolism or membrane fluidity. The 2H from C-18 of stearic acid is dominantly located at the original position of the fatty acid in the 2H-labeled membrane vesicles, as studied by a tracer experiment with [1-14C]stearic acid. We found that three to four 2H-labeled species were present at 19 degrees C in 2H NMR spectra of the 2H-labeled membrane vesicles prepared from B16 melanoma cells. The extent of peak-splittings due to 2H-quadrupole interaction decreased as the temperature rose, and a definite point of phase transition was not observed. At elevated temperature, 2H-labeled lipids undergo fast exchange between the bilayer and an isotropic phase such as oil phase of triolein or inverted micelles in lipid polymorphs. We further analyzed the change of membrane organization in mouse B16 melanoma cells treated with 12-O-tetradecanoylphorbol-13-acetate (TPA), which strongly inhibited melanogenesis. The magnitude of the quadrupole splitting at 19 degrees C in membranes from TPA-treated cells was significantly less (40%) than in the untreated control. This is mainly explained by decreased molecular ordering (fluidity) due to the increased amount of unsaturated fatty acids in the membranes of TPA-treated cells.     

The structure and conformation of a water-insoluble (1-->3)-,(1-->6)-beta-d-glucan from the fruiting bodies of Pleurotus florida

A water-insoluble glucan, PFPSIN, has been isolated from the aqueous extract of an edible mushroom Pleurotus florida. On the basis of total acid hydrolysis, methylation analysis, periodate oxidation, Smith degradation, and (13)C NMR experiments, the repeating unit of the polysaccharide was established as Conformational analysis revealed the triple helical conformation of this glucan.     

Cellular structure and function of mouse adrenocortical tumor cells Y-1 in the post-treatment state of low Ca2+

Under lowered Ca2+ content and in the post-treatment state of low Ca2+, we studied the cellular structure and functioning in mouse adrenocortical tumor cells, Y-1. These cells had been maintained in Ham F12 medium containing 10% fetal calf serum. The Ca2+ present in this complete medium was 0.39 mM. Under a slightly lowered Ca2+ (0.29 mM) produced by EGTA, the cells had many blebs on their surfaces and specific functional activity decreased in steroidogenesis as did ACTH reactivity. In the post-treatment state of a low Ca2+, the cellular surface was covered with many short microvilli and there was greater cellular activity than in the control cells. When the Ca2+ concentration was below 0.17 mM, the cellular structure and functioning were disturbed, and there was no recovery even at the physiological Ca2+ after the removal of EGTA.