摘要:

AbstractThe occurrence of the endogenous regulatory response to high rates of 2‐deoxyglucose (2‐DG) uptake, as previously described for C6 glioma cells during incubation with 2 mM 2‐DG (Lange et al.: J. Cell. Physiol., 1989), was studied in 3T3‐L1 preadipocytes and adipocytes, and the influence of insulin on this endogenous uptake regulation was examined. In contrast to 3T3‐L1 preadipocytes, insulin‐sensitive differentiated 3T3‐L1 adipocytes displayed the time‐dependent cyclic pattern of 2‐DG uptake rates characteristic of the membrane‐limited and endogenously regulated cellular state of hexose utilization. Although insulin induced a threefold stimulation of 2‐DG tracer uptake in adipocytes, the hormone did not additionally stimulate the uptake rates or affect the periodic response: maximum and minimum levels of uptake remained unchanged. Scanning electron microscopy (SEM) revealed that the acquirement of the differentiated state is accompanied by a conspicuous transformation of the smooth surface of undifferentiated 3T3‐L1 cells into a surface covered by numerous microvilii of uniform size and appearance. Treatment with insulin (10 mU/ml; 10 minutes) converted these microvilii into voluminous saccular membrane protrusions of the same type as had been formed during incubation of 3T3‐L1 adipocytes with 2 mM 2‐DG, and which have previously been shown to be involved in the endogenous uptake regulation of C6 glioma cells (Lange et al.: J. Cell. Physiol., 1989). These insulin‐induced saccated membrane areas appeared to become integrated into the cell surface. Accordingly, insulin treatment caused a twofold increase of the intracellular distribution space of 3‐O‐methylglucose (3‐OMG) in 3T3‐L1 adipocytes. This insulin‐induced increase of the 3‐OMG distribution space exhibited the same time (t1/2 = 2–2.5 minutes) and dose dependence (EC50 = 20 nM) as the insulin‐induced stimulation of 3‐OMG transport. Glucose deprivation during the differentiation period inhibited the outgrowth of microvilii from the cell surface. Glucose starvation (18 hours at<0.5 mM) induced a conspicuous reduction of the length of microvilii on differentiated 3T3‐L1 cells. In this state, the stalks of the microvilii are almost invisible and the enlarged spherical tips of the microvilii (with an average diameter of 370 nm compared to 230 nm of fed cells) appeared to protrude directly out of the cell surface. Starvation‐induced shortening of microvilli was accompanied by a threefold increase of the basal 3‐OMG transport rate and a greater than twofold increase of the intracellular 3‐OMG distribution space as compared to fed cells (10 mM; 18 hours). Starved cells showed nearly the same transport rates and 3‐OMG distribution spaces as insulin‐treated fed cells. 3‐OMG uptake by unstimulated 3T3‐L1 adipocytes, preincubated for 2 hours in glucose‐free medium, displayed a biphasic time course exhibiting high rates during the first 5–10 seconds followed by a constant low rate for the next 20–25 seconds. These results are compatible with the idea of a small cellular entrance compartment for hexose transport, as previously formulated for C6 glioma cells (Lange et al.: J. Cell. Physiol., 1989), which is formed by the internal space of surface protrusions such as microvilli and lamellae and which is separated from the cellular main compartment by a diffusion barrier. In 3T3‐L1 adipocytes, insuliti appeared to enlarge this entrance compartment, thereby reducing the effectiveness of the diffusion barrier. Both the endogenous mechanism of uptake regulation and the down‐regulation of transport by high glucose feeding (glucose‐curb) are supposed to control the rate of hexose uptake in a similar way by modulating the diffusional

ISSN:0021-9541    

DOI:10.1002/jcp.1041420102

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1990

数据来源: WILEY

摘要:

AbstractWe have examined conditioned medium (CM) from cultures of normal rat mammary epithelial (RME) cells for growth factor activity on fresh RME cell cultures. RME cell‐derived CM contained potent growth inhibitory activity toward fresh RME cell cultures when the medium was acidified by dialysis against 1% acetic acid prior to concentration. Dialysis of the CM at neutral pH resulted in CM that had growth stimulatory activity and no inhibitory activity. The acid‐activated growth inhibitor was heat and acid stable, protease sensitive, and eluted from a Bio‐Gel p60 column with a peak of activity in the 28 kDa range. Incubation of the acidified‐concentrated CM with neutralizing antiserum (affinity purified IgG) against transforming growth factor (TGF)‐β completely abolished the inhibitory activity of the CM. Furthermore, RME cell growth in the presence of the growth inhibitor plus TGF‐β antiserum was greater than that observed in growth medium alone. Subsequent experiments demonstrated that addition of TGF‐β antiserum alone to serum‐free medium enhanced RME cell growth, whereas addition of nonimmune IgG was without effect even at 25‐fold higher concentrations. Zymographic analysis of RME‐CM revealed the presence of plasminogen activator proteases that may mediate the partial activation of the latent growth factor. These results indicate that normal RME cells secrete a latent TGF‐β‐like growth factor into conditioned medium. Furthermore, the results indicate that some of the latent growth factor is activatedin situand contributes to the growth potential of the cells in primary cul

ISSN:0021-9541    

DOI:10.1002/jcp.1041420103

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1990

数据来源: WILEY

摘要:

AbstractHamster tracheal epithelial (HTE) cells maintained in primary culture show the induction of specific keratin species under vitamin A‐deficient conditions. A comparison was made between the morphology and the expression of keratins in HTE cells in vivo and in primary culture with and without vitamin A. HTE cells cultured in serum‐free, vitamin A‐supplemented medium formed a simple cuboidal, ciliated monolayer and produced four simple epithelial keratins (7, 8,18, and 19). In contrast, vitamin A‐deficient HTE cells, which were squamous‐like and stratified in culture, produced a more complex keratin pattern, with the induction of four additional keratin species (5,6, 14, and 17). A keratin pair whose expression serves as a marker of stratified epithelia was induced, as well as a single keratin species unique to lesions of squamous metaplasia in vitamin A‐deficient hamster tracheal organ cultures. Thus it appears that HTE cells retain the ability to respond to a deficiency in vitamin A through squamous differentiation and increased keratin production when removed from the intact organ and maintained in primary culture in a chemically defined medium. This system may be useful for the study of mechanisms underlying the squamous differentiation of respiratory epithelial cells in the development of bronchog

ISSN:0021-9541    

DOI:10.1002/jcp.1041420104

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1990

数据来源: WILEY

摘要:

AbstractWe have examined the effects of in vitro aging on the growth capacity of human umbilical vein endothelial cells (HUVECs) under the influence of tumor necrosis factor (TNF) with or without interferon‐γ (IFN‐γ). The growth and colony‐forming abilities of control cells were impaired with advancing age in vitro, especially at later stages (more than 70–80% of life span completed). It was found that treatment with TNF inhibited growth and colony‐forming efficiency at any in vitro age. The effects of TNF were shown to increase with increasing in vitro age, as reflected by a more pronounced increase in doubling times, a decrease in saturation density, and a reduction in colony‐forming efficiency. However, the characteristics of TNF receptors, including the dissociation constant, and the number of TNF‐binding sites per cell‐surface area remained rather constant. The effect of TNF was augmented by IFN‐γ at a dose that alone affected growth and colony formation only slightly. The augmentation by IFN‐γ was also found to depend on in vitro age; the synergy with TNF in the deterioration of colony‐forming ability was observed only in “aged” cells. These results suggest that the intrinsic responsiveness of HUVECs to growth‐inhibiting factors, as well as to growth‐stimulating facto

ISSN:0021-9541    

DOI:10.1002/jcp.1041420105

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1990

数据来源: WILEY

摘要:

AbstractThe effect of pertussis toxin (PT) on transforming growth factor β1 (TGFβl)‐induced proto‐oncogene expression was investigated in AKR‐2B fibroblasts. PT substantially abolished c‐sis and c‐myc mRNA expression following TGFβl stimulation. This inhibitory effect was specific for TGFβ1‐stimulated proto‐oncogene expression and associated with the ADP‐ribosylation of a 41‐kDa substrate. Actinomycin D decay and nuclear run‐on experiments demonstrated that the inhibitory effects of PT are a result of decreased transcriptional activation and not to an increased decay of proto‐oncogene message. PT did not, however, affect TGFβl‐stimulated fibronectin and collagen mRNA accumulation nor did it have any inhibitory effect on TGFβl‐induced morphological transformation. These data indicate that TGFβl‐stimulated gene expression is coupled to multiple pathways distin

ISSN:0021-9541    

DOI:10.1002/jcp.1041420106

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1990

数据来源: WILEY

摘要:

AbstractWhen dispersed chief cells from guinea pig stomach were first incubated with carbachol, washed, and then reincubated with carbachol in fresh incubation solution, the stimulation of pepsinogen secretion and the rise in intracellular calcium concentration during the second incubation were reduced. Carbachol did not cause residual enzyme secretion, but the same range of concentrations that causes enzyme secretion caused desensitization that was rapid, temperature dependent, and reversible with time. Preincubation with carbachol caused approximately a 65% reduction in enzyme secretion stimulated during a subsequent incubation with this agonist, but the potency of carbachol was unaffected. Prior exposure to carbachol also reduced subsequent stimulation caused by cholecystokinin (CCK‐8), gastrin I, ionophore A23187, or 12‐O‐tetradecanoylphorbol 13‐acetate but did not alter stimulation by any agonist that increases cellular cAMP. Carbachol pretreatment of Fura‐loaded chief cells caused a threefold increase in the EC50for carbachol‐stimulated [Ca2+]iand approximately a 30% reduction in the maximal rise in [Ca2+]iin response to carbachol or CCK‐8. Inhibition of [N‐methyl‐3H] scopolamine binding by carbachol following carbachol pretreatment indicated that modulation of receptor affinity or number did not account for functional desensitization. These data indicate that carbachol causes heterologous desensitization of pepsinogen secretion stimulated by agonists that mobilize cellular Ca2+or activate protein kinase C through a postreceptor action and suggest that an attenuated rise in chief cell calcium is one mechanism mediating the desensitization of

ISSN:0021-9541    

DOI:10.1002/jcp.1041420107

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1990

数据来源: WILEY

摘要:

AbstractThe effect of the phorbol ester tumor promoter 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) on cell invasion was studied using an in vitro assay for cell invasion through a reconstituted basement membrane matrix (Matrigel). TPA inhibited the invasiveness of malignant human fibrosarcoma HT1080 cells. In contrast, WI‐38 lung fibroblasts, which show a very low invasive capacity, were stimulated (3‐fold) to invade Matrigel after exposure to TPA for 48 hours. The inhibitory or stimulatory effects of TPA on cell invasion were correlated with a decrease or an increase in cell motility and collagenase IV activity, respectively. Synthetic diacylglycerols partially mimicked the inhibitory action of TPA on HT1080 cells but failed to stimulate WI‐38 cell invasion. Immunoblots demonstrated that in both cell lines the α and β isoforms of protein kinase C were equally down‐regulated after a 5 hour exposure to TPA despite the basal low level of protein kinase C polypeptide in the malignant cells. Thus, whereas in WI‐38 cells induction of an invasive behavior could be observed in the absence of protein kinase C, in the malignant cells disappearance of the kinase was associated with a non

ISSN:0021-9541    

DOI:10.1002/jcp.1041420108

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1990

数据来源: WILEY

摘要:

AbstractCells isolated from fetal rat calvaria (RC) and maintained in vitro in medium containing ascorbic acid and B‐glycerophosphate form three‐dimensional, mineralized nodules having the histological, immunohistological, and ultrastructural characteristics of woven bone. We have studied the effects of forskolin (FSK), a diterpene that activates adenylate cyclase, in this system. While 10−7−10−5M FSK significantly stimulated cAMP levels in RC cells, lower concentrations did not. cAMP levels with 10−5M FSK reached a maximum by 30 min at 37°C and returned to basal level in 2‐3 hr. Changes in cAMP levels correlated with changes in cellular shape: cells treated with 10−5M FSK assumed a stellate morphology, lost microfilament bundles, and reduced their substrate adhesiveness, while cells treated with 10−9M were not affected. Exponential growth and saturation densities of FSK‐treated cultures were similar to untreated cultures, indicating that FSK was neither toxic nor stimulatory to the population. The effect on bone nodule formation of FSK present continuously depended on concentration: 10−5M FSK significantly inhibited the number of nodules formed, while 10−9M FSK significantly stimulated bone nodule formation. Single short treatments with either 10−5M or 10−9M FSK had no effect on nodule formation, but repeated short duration treatments (1 hr every 2 days for 21 days) gave results similar to continuous exposure. These results indicate that intermittent elevations in intracellular cAMP have an inhibitory effect on bone formation. In addition, our work indicates that low concentrations of FSK stimulate differentiation of osteoprogenitor cells possibly through

ISSN:0021-9541    

DOI:10.1002/jcp.1041420109

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1990

数据来源: WILEY

摘要:

AbstractThe human squamous cell carcinoma SqC/Y1 undergoes spontaneous terminal differentiation in the confluent state. The degree of maturation was markedly increased by glucocorticoids and by both human recombinant and placental lipocortin I. Western analyses demonstrated cellular secretion of lipocortin into the medium. Glucocorticoid‐induced maturation was antagonized by a lipocortin l‐specific monoclonal antibody, by phospholipase A2(PLA2), and by arachidonic acid. Induction of the differentiation of SqCC/Y1 cells by lipocortin I was prevented by arachidonic acid. The PLA2 inhibitor, dibromoacetophenone, caused an increase in envelope‐competent cells indicating that inhibition of PLA2 results in induction of differentiation. Epidermal growth factor prevented the induction of differentiation by both lipocortin I and by glucocorticoids. The nonsteroidal lipoxygenase/cyclo‐oxygenase inhibitor, phenidone, also increased SqCC/Y1 differentiation, suggesting that leukotrienes, thromboxanes, and/or prostaglandins may be involved in lipocortin‐mediated regulation of SqCC/Y1 maturation. The findings support a role for lipocortin I in mediating the effects of glucocorticoids on epidermal cell differ

ISSN:0021-9541    

DOI:10.1002/jcp.1041420110

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1990

数据来源: WILEY

摘要:

AbstractRelease of calcium from intracellular stores of rat basophilic leukemia cells was monitored using the fluorescent probe chlortetracycline. The ability of chlortet‐racycline to indicate release from intracellular calcium stores was initially validated. The decrease of chlortetracycline fluorescence upon antigen‐stimulation was not the result of secretion of granule‐associated dye or of changes in the properties of the membranes. The chlortetracycline fluorescence signal was not influenced by Ca2+influx across the plasma membrane. Results obtained from these chlortetracycline fluorescence measurements corresponded well with45Ca efflux data, an indirect measurement of release of calcium from stores. Chlortet‐racycline was used to examine the rate of antigen‐induced release of calcium from stores, the depletion of intracellular calcium stores by EGTA, and the relationship between the antigen‐stimulated release of stored calcium and exocytosis. Chlortetracycline was shown to be a useful qualitative indicator for the release of intracellular calcium with a relatively rapid re

ISSN:0021-9541    

DOI:10.1002/jcp.1041420111

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1990

数据来源: WILEY