Changes in cytoplasmic and lysosomal enzyme activities in cultured rat heart cells: the relationship to cell differentiation and cell population in culture

Postnatal rat heart cells in culture enriched with respect to muscle cells were obtained by either high density seeding or by the replating technique. [3H]Thymidine incorporation to DNA and the enzymatic pattern of cytoplasmic and lysosomal enzymes have been studied as a function of the culture's age, of seeding density, and replating. It was shown that replating maintains predominance of myocyte population for at least 2 wk in culture; heavy seeding density allows homogeneous myocyte population for the 1st wk in culture; and the enzyme profile of the culture may serve as an indicator for the type of cell population in culture and its state of differentiation.     

Changes of lysosomal proteinase activities and their expression in rat cultured keratinocytes during differentiation

The cathepsins B, H and L, lysosomal cysteine proteinases, play a major role in intracellular protein degradation. These proteinase activities and expressions were examined in a Ca2+ regulated epidermal culture system which consists of two morphological cell types: undifferentiated cells grown in low Ca2+ (0.1 mM concentration) and differentiated cells grown in high Ca2+ (1.8 mM concentration), respectively. Cathepsin B and L activities of the differentiated cells showed a several-fold increase compared to that of the undifferentiated cells. In addition, by using CM-cellulose column chromatography, cathepsin B and L were separated and the level of cathepsin L activity increased significantly. Cathepsin B, L and H were also detected by using an immunoblotting procedure in which their bands were expressed after differentiation was induced by the increasing calcium concentration. Cathepsin L activity and immunostaining intensity reached a maximum at 1 or 2 days of differentiation. In contrast, cystatin alpha (an endogenous inhibitor of cysteine-dependent cathepsins) appeared in the final stage of differentiation. These results indicate that the expression of epidermal cathepsins and their endogenous inhibitor are involved in part of the program of cell differentiation and the terminal differentiation process in cultured rat keratinocytes.     

Interactions of nitric oxide and oxygen in cytotoxicity: Proliferation and antioxidant enzyme activities of endothelial cells in culture

Nitric oxide (NO) shows cytotoxicity, and its reaction products with reactive oxygen species, such as peroxynitrite, are potentially more toxic. To examine the role of O₂ in the NO toxicity, we have examined the proliferation of cultured human umbilical vein endothelial cells in the presence or absence of NO donor, ((Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)-amino]diazen-1-ium-1,2-diolate) (DETA-NONOate) (100–500 μM), under normoxia (air), hypoxia (< 0.04% O₂) or hyperoxia (88–94% O₂). It was found that the dose dependency on NONOate was little affected by the ambient O₂ concentration, showing no apparent synergism between the two treatments. We have also examined the effects of exogenous NO under normoxia and hyperoxia on the cellular activities of antioxidant enzymes involved in the H₂O₂ elimination, since many of them are known to be inhibited by NO or peroxynitrite in vitro. Under normoxia DETA-NONOate (500 μM) caused 25% decrease in catalase activity and 30% increases in glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities in 24 h. Under hyperoxia NO caused about 25% decreases in activities of catalase, glutathione reductase and glucose-6-phosphate dehydrogenase. The H₂O₂ removal rate by NO-treated cells was computed on the mathematical model for the enzyme system. It was concluded that the cellular antioxidant function is little affected by NO under normoxia but that it is partially impaired when the cells are exposed to NO under hyperoxia.     

Changes in some chromatin and cytoplasmic enzymes of perinatal rat hepatocytes during culture

Summary Hepatocytes prepared from rats at various perinatal stages were cultured in selective medium that does not allow fibroblastic cell growth. Cell population remained homogeneous during the culture. Hepatocytes undergo divisions for a period, which varies according to the stage of development of the rat. Light and electron microscope observations showed the presence of numerous cytoplasmic organelles; moreover, hydrocortisone-induced structures similar to bile canaliculi. Chromatin protein kinase decreased rapidly during culture except in samples prepared from 17-day fetuses in which it remained unchanged for 2 days and decreased to a lesser extent afterwards. Chromatin nonhistone proteins were incubated with (γ-³²P) ATP and the phosphorylation pattern analyzed on polyacrylamide gels. Many radioactive peaks were observed in chromatin proteins from 17-day fetuses; they were much lower in proteins from 19-day fetuses. The phosphorylation pattern was analyzed in hepatocytes after 2 days of culture. Many radioactive peaks were observed with proteins from heapatocytes taken from 17-day fetuses; no radioactivity was observed in proteins from 19-day fetuses. This is in contrast with the absence of radioactive peaks in chromatin proteins from adult rat hepatocytes. In cytoplasm, aldolase and pyruvate kinase specific activities varied according to the age of the rat. They strongly decreased during culture except in hepatocytes from 15-and 17-day fetuses, in which they remained stable for at least 5 days. The stability of chromatin and cytoplasmic enzymes in hepatocytes from 17-day fetuses could result from their ability to be regulated by hormones that are secreted at this stage of development.     

Ischemic myocardial injury in cultured heart cells: Leakage of cytoplasmic enzymes from injured cells

Summary An in vitro model of myocardial ischemia has been established with primary monolayer cultures of postnatal rat myocardial cells. Ischemic conditions were simulated in vitro by subjecting the myocardial cell cultures to various levels of oxygen and glucose deprivation. The experimental protocol consisted of treatment with 20% or 0% O₂ and 1000, 500 or 0 mg glucose per 1 of medium for 4 or 24 hr. Control cultures were treated with 20% O₂ and 1000 mg glucose. After the ischemic treatments, cultures of beating muscle (M) cells were evaluated for signs of injury, i.e. leakage of cytoplasmic enzymes into the culture medium. Differences were found in leakage of lactate dehydrogenase (LDH) and creatine phosphokinase (CPK) from the cultures that were exposed to partial ischemia of glucose deprivation and from those cultures that were exposed to total ischemia of oxygen and glucose deprivation. Glucose deprivation alone resulted in a slight-to-moderate loss of LDH and CPK from the cells, whereas total ischemia resulted in a significant release of the two cytoplasmic enzymes. When the cultures were allowed to recover after ischemic treatment in complete medium (1000 mg glucose) and a normal atmosphere of 20% O₂, they had levels of LDH leakage comparable to those of control cultures. Cell viability and total protein content of the ischemic cultures did not differ significantly from controls. This study was supported by Research Grant HL 18647 from the National Heart, Lung, and Blood Institute.     

Changes in ultrastructures and enzyme activities during differentiation of myeloid leukemia cells to normal macrophages

Changes in ultrastructures and in enzyme activities were investigated electron microscopically, cytochemically and biochemically when mouse myeloid leukemia cells, Ml cell line, successfully differentiated to normal macrophages after incubation with a conditioned medium harvested from secondary embryo fibroblasts, or a lipopolysaccharide from Salmonella typhosa. The number of mitochondria increased significantly accompanied by the enhanced activity of cytochrome oxidase per cell, although the activity in each mitochondrion remained unchanged. The rough-surfaced endoplasmic reticulum elongated and often exhibited a concentrically multilayered lamellae. Glucose-6-phosphatase activity, a marker enzyme for the endoplasmic reticulum, also increased. Primary lysosomes were newly formed where acid phosphatase activity was positively demonstrated. Ten-nm cytoplasmic microfilaments, mainly forming bundles, and other microfilaments less than 6 nm wide were formed newly and abundant. Budding of type C viruses from the plasma membranes was reduced strikingly. Another established cell line, Mm-1, which spontaneously differentiated from the Ml cell line, was characterized completely by a macrophage, in which azurophilic granules (primary lysosomes), secondary lysosomes possessing strong activity of acid phosphatase and 10-nm microfilaments were most remarkable. These non-transplantable Mm-1 cells sometimes exhibited budding of viruses.     

Heme-biosynthetic enzyme activities and porphyrin accumulation in normal liver and hepatoma cell lines of rat

The activities of four heme-biosynthetic enzymes, -aminolevulinic acid (ALA) synthase, ALA dehydratase, porphobilogen (PBG) dearninase, and ferrochelatase, were studied in five epithelial cell lines of normal rat liver origin (Re, REC-10, RLC-24, M, Culb-TC) and five cell lines derived from Yoshida ascites hepatoma (JTC-1, JTC-2, JTC-15, JTC-16, JTC-24). The JTC series of hepatoma-derived cell lines exhibited decreased ALA synthase activity and increased ALA dehydratase activity, although the activities of all four enzymes and the Km values for their respective substrates varied widely from one cell line to another, a finding suggesting that specific regulatory mechanisms for porphyrin metabolism might operate in each cell type. M cells, which were transformed by 4-dimethylaminoazobenzene in vitro, gave the most abnormal Km values of heme-biosynthetic enzymes among all the cell lines studies, and were found to accumu2ate hematoporphyrin derivative (HpD).Abbreviations ALA o-aminolevulinic acid - DAB 4-dimethyl aminoazobenzene - HpD hematoporphyrin derivative - 4NQO 4-nitroquinoline 1-oxide - PBG porphobilinogen     

Lysosomal enzyme activities and RNA turnover rates in growing and nongrowing WI-38 and HeLa cells

Summary Activities of three lysosomal enzymes—acid RNase,N-acetyl-β-D-glucosaminidase and acid phosphatase—were determined during the growth cycles of WI-38 and HeLa cells, as well as in radiation-arrested WI-38 cells. In confluent and growth-arrested cultures of WI-38 cells, the lysosomal RNase increased six- to sevenfold; glucosaminidase, four- to five-fold; and phosphatase, two- to threefold. In HeLa cells, the lysosomal enzymes also increased in confluent cultures, but less than twofold; and the RNase level increased only transiently. In both WI-38 and HeLa cells, the rate of RNA breakdown, also increased as cultured approached confluency. The rate of turnover of RNA, like the level of acid RNase, was higher in WI-38 cells than in HeLa cells (4 d half-life compared to 8 d). The increase in acid RNase could be prevented by incubation of cells in NH₄Cl, but the rate of turnover in the presence of NH₄Cl increased just as much when cells became confluent or stopped growth. The content of acid RNase could be changed more than 10-fold without altering the rate of RNA turnover. It is suggested that the increase in enzyme level is more important for possible autophagy or increased digestion of engulfed RNA, rather than for normal RNA turnover, when growth stops. This study was supported by Grant GM-21357 from the National Institutes of Health.     

Rat Sertoli cell aromatase cytochrome P450: Regulation by cell culture conditions and relationship to the state of cell differentiation

Summary Primary cultures of immature rat Sertoli cells in plastic dishes are highly responsive to follicle stimulating hormone (FSH) and its second messenger, cAMP, in metabolizing testosterone to estradiol, thus indicating the presence of an active, hormone-regulated aromatase cytochrome P450 (P450arom). However, in vivo studies indicated that P450arom is FSH-responsive only in very young animals, where the cells have not yet differentiated, but they lose this ability later on in development. Sertoli cells grown on Matrigel (a reconstituted basement membrane), laminin (a basement membrane component), or in bicameral chambers coated with Matrigel, assume structural and functional characteristics more similar to that of in vivo differentiated Sertoli cells. When the cells were cultured on laminin or Matrigel, the FSH- and cAMP-induced estradiol production was greatly reduced by 30 and 60%, respectively. When Sertoli cells were cultured in bicameral chambers coated with Matrigel, no induction of testosterone aromatization by FSH or cAMP was observed. However, FSH-induced cAMP formation was greater when the cells were cultured on basement membrane or in the chambers than on plastic dishes. These results suggest that culture conditions favoring the assumption by Sertoli cells of a phenotype closer that of the differentiated cells in vivo (tall columnar and highly polarized) suppress the induction of P450arom by FSH and cAMP. We then examined the mechanism(s) by which cell phenotype affects p450arom activity. Northern blot analyses of Sertoli cell RNA revealed one major band of 1.9 Kb and two minor bands of 3.3 and 5.2 Kb. However, there were no changes at the level of the expression of P450arom messenger RNA under the different culture conditions. No differences were found in P450arom enzymatic activity measured by the³H₂O release method in microsomes prepared from Sertoli cells cultured under the various conditions. Similarly, no differences were observed in the amount of protein detected by immunoblot analysis of Sertoli cell extracts using an antiserum raised against the human placental enzyme. Recombination experiments using microsomes from cells cultured on plastic or in the chambers and cytosol from control or FSH-treated cells cultured on plastic also proved inadequate in inducing P450arom activity. These data suggest that: a) P450arom activity could be used as a specific marker for Sertoli cell differentiation, and b) the differentiation process in Sertoli cells is associated with specific changes in the microenvironment or the regulation of P450arom, or both, that rendered the enzyme insensitive to FSH or cAMP induction.     

Antioxidant enzyme activities and lipid peroxidation induced by eicosapentaenoic acid (EPA) in PC12 cells

Eicosapentaenoic acid (EPA) is one of the major dietary polyunsaturated fatty acids and induces apoptosis in several cancer cells. In this study, the EPA induced lipid peroxidation and response of antioxidative enzymes have been investigated in rat pheochromocytoma PC12 cells to elucidate the mechanisms of apoptosis induced by the polyunsaturated fatty acid EPA. We have analyzed superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPX) activities and glutathione (GSH) contents in PC12 cells after exposure to different concentrations of EPA. Lipid peroxidation was shown to increase in the presence of EPA as an indication of the oxidative damage. Lipid peroxidation was enhanced by EPA in a dose-dependent manner, and the loss of cell viability was partially reversed by vitamin E. In the case of antioxidant enzyme activities, SOD and GPX activities and GSH contents increased significantly at 50 μmol/L EPA and were respectively 2.41-fold (p < 0.01), 3.49-fold (p < 0.05), and 1.43-fold (p < 0.05) higher than controls. The CAT activity at 10 μmol/L had the highest value and was increased by 25.83% (p < 0.05) compared to control. The results suggest that in PC12 cells the mechanism of apoptosis induced by EPA may be partly due to lipid peroxidation.     

Mitral cell differentiation and synaptogenesis of rat presumptive olfactory bulb in organ culture

Summary The ultrastructure of differentiating rat presumptive olfactory bulb in organ culture was investigated with particular reference to mitral cell differentiation and formation of synapses. The presumptive olfactory bulb and olfactory mucosa were dissected en bloc from rat embryos on the fifteenth day of gestation and cultured for 7 days, after which the expiants were examined by electron microscopy. The presumptive olfactory bulb had differentiated into a laminated structure with layers corresponding to the glomerular, external plexiform and mitral cell layers. Mitral-like cells were identified by their location and large cell size. Ultrastructural observations indicated that they were relatively well-differentiated. Their dendrites extended into the glomerular layer in which they were postsynaptic to incoming olfactory axons. The distal part of these dendrites frequently contained coated vesicles. Both asymmetrical and symmetrical synapses were found. The symmetrical synapses involved dendrodendritic contacts between periglomerular cells. Synapses in reciprocal arrangements were not observed in the organ cultures.     

鼠骨髓间充质干细胞的分离培养及定向诱导分化为内皮样细胞

目的：探讨体外大鼠骨髓间充质干细胞（rBMMSCs）的分离培养和血管内皮生长因子（VEGF）、碱性成纤维细胞生长因子（bFGF）对其定向诱导为内皮样细胞（ELCs）的可行性。方法：采用Percoll（1.073g/ml）分离液分离骨髓单个核细胞,用含10%胎牛血清（FBS）的LG-DMEM培养基贴壁纯化培养,倒置显微镜、免疫细胞化学法、流式细胞仪、MTT法、透射电镜（TEM）联合对rBMMSCs形态、表型、生长曲线、细胞周期以及超微结构进行鉴定;诱导后的细胞,采用倒置显微镜观察细胞形态,免疫细胞化学法检测CD31、CD144（VE-cadherin）和CD34表达以及摄取Dil-ac-LDL、结合FITC-UEA-1的功能特点。结果：rBMMSCs呈长梭形,漩涡状排列。细胞生长曲线显示潜伏期、对数生长期和平台期,符合干细胞的生长规律。透射电镜结果表明：rBMMSCs有两种不同的形态结构,其中体积较小、核质比大、胞质内细胞器稀少者为处于未分化或分化较低状态的幼稚型rBMMSCs。细胞周期分析显示：第4代细胞G0/G1期为95.67%,表明绝大部分细胞处于非增殖状态;诱导后的部分细胞形态可见类似ELCs改变,表达血管内皮细胞（ECs）特异性表面标志CD31、CD34和CD144,具有摄取Dil-ac-LDL以及结合FITC-UEA-1的功能特点。结论：采用Percoll密度梯度离心与贴壁培养相结合的方法所培养的rBMMSCs在体外具有定向诱导分化为ELCs的潜能,可能成为血管组织工程理想的种子细胞来源。     

Modulation of lysosomal enzyme levels in cultured cells: effects of alterations in cell density, balanced growth, and endocytosis

Factors which influence lysosomal enzyme accumulation in cultured cells have been studied. In cell types of both fibroblast (3T6) and epithelial (HeLa) origin, acid phosphatase and β-N-acetylglucosaminidase activities increase with increasing cell density. However, in other cell lines such as BHK or chick embryo fibroblasts, little or no accumulation of lysosomal enzymes occurred with increased cell density. Increased lysosomal enzyme activity need not necessarily be accompanied by alterations in rate of cell growth, rate of pinocytosis, or amount of internalized degradable macromolecules. The stimulus for lysosomal enzyme accumulation appears to require cell contact, since sparsely plated cells do not exhibit lysosomal enzyme accumulation. In 3T6 cells, lysosomal enzymes also accumulate during “step-down” conditions, such as amino acid or serum depletion, or during unbalanced growth resulting from inhibition of cytokinesis or DNA synthesis. Increases in the specific activity of lysosomal enzymes which occur during step-down conditions or unbalanced growth require cell contact, since they are not seen in sparse cells, but are observed in medium- and high-density cells incubated in serum-free medium. Studies employing actinomycin D suggest that lysosomal enzyme levels are regulated primarily via control of enzyme synthesis, rather than enzyme degradation.     

Transient stress and stress enzyme responses have practical impacts on parameters of embryo development, from IVF to directed differentiation of stem cells

In this review, we discuss the expression, regulation, downstream mechanisms, and function of stress-induced stress enzymes in mammalian oocytes, peri-implantation embryos, and the stem cells derived from those embryos. Recent reports suggest that stress enzymes mediate developmental functions during early mammalian development, in addition to the homeostatic functions shared with somatic cells. Stress-induced enzymes appear to insure that necessary developmental events occur: many of these events may occur at a slower rate, although some may occur more rapidly. Developmental events induced by stress may be mediated by a single dominant enzyme, but there are examples of responses that require the integration of more than one stress enzyme. The discussion focuses on the consequences of stress as a function of duration and magnitude, and this includes an emerging understanding of the threshold levels of duration and magnitude that lead to pathology. Other topics discussed are the reversibility of the developmental as well as homeostatic consequences of stress, the further problems with readaptation after stress subsides, and the mechanisms and functions of stress enzymes during early mammalian development. The analyses are done with specific concern for their practical impact in assisted reproductive technology (ART) and stem cell technologies.     

Serum-free,chemically defined medium to evaluate the direct effects of growth factors and inhibitors on proliferation and function of neonatal rat cardiac muscle cells in culture

Summary Neonatal rat cardiac myocytes were isolated and cultured to evaluate the effects of growth factors and inhibitors on proliferation, survival, and functions in a serum-free medium. Insulin and transferrin in MCDB 107 nutrient medium elicited DNA and protein synthesis in cells on a fibronectin-coated culture surface in serum-free medium. Insulin was most effective on both DNA and protein synthesis in serum-free culture conditions. The serum-free, hormone-supplemented medium eliminated the contamination of noncardiac myocytes and supported the long-term survival (over 18 d) of cardiac myocytes. Dexamethasone was required to induce optimal contractility with or without insulin and transferrin. Serum contained both negative and positive effectors of DNA and protein synthesis of the cardiac myocytes. Concentrations of serum (above 5%) inhibited DNA and protein synthesis. Low density lipoprotein (LDL) accounted in part for the inhibitory activity. The serum-free culture system provides a useful model to elucidate the role of hormones, growth factors, and drugs in heart cell regeneration and function.     

Failure of insulin cells to develop in cultured embryonic chick pancreas: A model system for the detection of factors supporting insulin cell differentiation

Summary Little being known about factors necessary for insulin cell differentiation, we tested the chance observation that these cells were virtually absent from collagen gel cultures of embryonic avian pancreas in which the other pancreatic endocrine cells were numerous. Five-day dorsal buds stripped of their enveloping mesenchyme were embedded in gel and overlaid by a defined medium containing serum, then cultured for 7 days. Immunocytochemical evaluation showed a very low proportion of insulin cells. Substitution of the gel by a polyamino acid coating slightly increased the proportion. In an attempt to test for ability of insulin cell formation to recover, we transferred explants first cultured in collagen gel to polyamino-acid-coated dishes for a further 7 days. No improvement resulted. In controls grown for 14 days on a polyamino acid coating, insulin cells disappeared completely. We conclude that collagen gel does not support survival and differentiation of chick embryonic insulin cells and that the medium used is lacking in some essential factor(s). Determination of their identity should prove possible by exploitation of this model.     

Drug-metabolizing enzyme activities in freshly isolated oval cells and in an established oval cell line from carcinogen-fed rats

The activities of several different phase I and phase II drug-metabolizing enzymes were measured in freshly isolated oval cells from rats fed a choline-deficient/DL-ethionine-supplemented diet for 6 weeks and alsoin vitro in the established oval cell line OC/CDE 6. No cytochrome P450 was spectrophotometrically measurable in both preparations and two cytochrome P450-dependent monoxygenase activities, aminopyrineN-demethylase and ethoxyresorufinO-deethylase, could not be detected in the oval cells of both sources. However, cytosolic glutathione transferase, microsomal expoxide hydrolase and UDP-glucuronosyltransferase activities were clearly measurable in oval cells. Similar enzyme activities were found in freshly isolated and cultured oval cells. The highest activities of these three enzymes were detected during the exponential growth phase of the cultured cells; thereafter the activities decreased until the cells reached confluency. Changes in phenol UDP-glucuronosyltransferase (UGT1A1) mRNA levels paralleled the variations in UDP-glucuronosyltransferase activity, i.e. they were high in exponentially growing oval cells and low in confluent cell cultures. Taking into account that oval cells are able to proliferate in the livers of rats continuously fed a choline-deficient/DL-ethionine-supplemented diet and that none of the analyzed drug metabolizing enzymes are involved in the activation or detoxication ofDL-ethionine, the described pattern might be part of a more general, nonspecific, protection mechanism enabling these cells to overcome the cytotoxic effects of a variety of carcinogens and to proliferate even in their presence. Furthermore, the expression of microsomal epoxide hydrolase, cytosolic glutathione transferase and UDP-glucuronosyltransferase appears to depend on the proliferative status of the cells.Abbreviations CDE choline-deficient/DL-ethionine-supplemented diet - GST glutathione transferase - mEH microsomal epoxide hydrolase - UGT UDP-glucuronosyltransferase     

Comparison of human-induced pluripotent stem cells and mesenchymal stem cell differentiation potential to insulin producing cells in 2D and 3D culture systems in vitro

Diabetes is one of the most common diseases in the world that is chronic, progressive, and costly, and causes many complications. Common drug therapies are not able to cure it, and pancreas transplantation is not responsive to the high number of patients. The production of the insulin producing cells (IPCs) from the stem cells in the laboratory and their transplantation to the patient's body is one of the most promising new approaches. In this study, the differentiation potential of the induced pluripotent stem cells (iPSCs) and mesenchymal stem cells (MSCs) into IPCs was compared to each other while cultured on poly(lactic-co-glycolic) acid (PLGA)/polyethylene glycol (PEG) nanofibrous scaffold as a 3D substrate and tissue culture polystyrene (TCPS) as a 2D substrate. Although the expression level of the insulin, Glut2 and pdx-1 genes in stem cells cultured on 3D substrate was significantly higher than the stem cells cultured on 2D substrate, the highest expression level of these genes was detected in the iPSCs cultured on PLGA-PEG. Insulin and C-peptide secretions from differentiated cells were also investigated and the results showed that secretions in cultured iPSCs on the PLGA-PEG were significantly higher than cultured iPSCs on the TCPS and cultured MSCs on both PLGA-PEG and TCPS. In addition, insulin protein was also expressed in the cultured iPSCs on the PLGA-PEG significantly higher than cultured MSCs on the PLGA-PEG. It can be concluded that differentiation potential of iPSCs into IPCs is significantly higher than human MSCs at both 2D and 3D culture systems.     

The relationship between cytotoxic effect and binding to mammalian cultured cells of Clostridium perfringens enterotoxin

Abstract The relationship between the cytotoxic effect and binding to different cell lines of Clostridium perfringens enterotoxin was investigated. The enterotoxin released ⁵¹Cr from Vero and MDCK cells labeled with Na₂-⁵¹CrO₄. The effect varied depending upon the dose of enterotoxin and the duration and temperature of the interaction. The enterotoxin gave no effect on FL, KB, or L-929 cells. [¹²⁵I]Enterotoxin bound specifically to Vero and MDCK cells via a binding site of distinct nature, but not to FL, KB, or L-929 cells. The number of the binding sites located on one MDCK cell (1.98 × 10⁶ sites/cell) was three times that on one Vero cell (5.64 × 10⁵ sites/cell), although the binding affinity of MDCK cell ( K _a/ 3.76 × 10⁷ M⁻¹) was 0.1 that of Vero cells ( K _a/ 3.23 × 10⁸ M⁻¹). Binding of the enterotoxin to susceptible cells was temperature-independent.