摘要:

AbstractWe studied the expression of osteoblastic markers in cultured cells isolated from the bone of 15 patients with different clinical forms of osteogenesis imperfecta (OI) and of seven fetal and postnatal controls. Cultured bone cells of ten OI patients produced abnormal collagen type I. Similar to controls, OI bone cells produced predominantly collagen type I with traces of collagen types III and V. The 1,25(OH)2vitamin D3‐stimulated synthesis of osteocalcin, a specific osteoblastic marker protein, was similar in OI bone cells and age‐matched controls. Bone cells from fetal controls and from patients with the perinatal lethal OI type II produced less osteocalcin than bone cells from postnatal controls and surviving OI patients. OI bone cells responded to parath.yroid hormone (PTH) by increased production of cAMP similar to controls. Bone cells from fetal controls and from OI type II donors showed a decreased response to PTH. Activity of the bone‐liver‐kidney isoenzyme alkaline phosphatase (AP) was detected in all control and OI bone cells. The expression of all osteoblastic markers was similar in bone cells producing abnormal collagen type I. These observations show that OI bone cells in vitro express a pattern of osteoblastic markers similar to age‐matched control bone cells indicating that osteoblastic differentiation is not altered by the underlying defects of collagen type I metabolism in OI bone cells. © 1993 Wiley

ISSN:0021-9541    

DOI:10.1002/jcp.1041570302

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

年代:1993

数据来源: WILEY

摘要:

AbstractWe investigated effects of bafilomycin A1, a specific inhibitor of vacuolar‐type H+‐ATPase (V‐ATPase), and its analogues on proliferation of various cultured cells. The proliferation of the various cell lines was suppressed by adding bafilomycin A1to the culture medium. This inhibitory effect appeared at a concentration of nanomolar order and was dose dependent. Although the suppression was reversible, the drug exerted not only suppression of the proliferation but also death to some cell lines. Drug concentration required for 50% inhibition of the cell proliferation during 48 h differed markedly depending on cell species and the sensitivity appears to increase by the transformation of the cells. Two derivatives of concanamycin A, an analogue of bafilomycin A1, also inhibited strongly V‐AT‐Pase in vitro and in vivo, and simultaneously cell proliferation. Two concanamycin A derivatives which have lost inhibitory effect on V‐ATPase lost inhibitory effect on cell proliferation as well. These results suggest that V‐ATPase is involved in the machinery maintaining the cell proliferation. © 1993 W

ISSN:0021-9541    

DOI:10.1002/jcp.1041570303

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

年代:1993

数据来源: WILEY

摘要:

AbstractTwo distinct mechanisms have been shown to mediate cytoplasmic pH (pHi) recovery in acid‐loaded peritoneal macrophages (Mψs): Na+/H+exchange and H+extrusion by vacuolar‐type (V‐type) H+ATPases. The present studies examined the relative roles of these two systems in maintaining pHiand cell function. Measurements of Mψ pHiand superoxide (O2−) production in response to stimulation with 12‐O‐tetradecanoyl phorbol 13‐acetate (TPA) were made at physiological or acidic extracellular pH (pHo) levels. The V‐type H+ATPase inhibitor, bafilomycin A1, and potent Na+/H+exchange inhibitor, N‐ethyl‐N‐propylamino amiloride (EPA), were used to examine the contributions of these ion transporters to pHiregulation and cell function. At pHo7.35, the complementary activities of the Na+/H+antiport and the V‐type H+ATPase mediate pHihomeostasis. At pHo6.7, maintenance of pHidepends primarily on H+ATPase activity: bafilomycin A1reduced pHifrom 6.8±0.02 in control cells to 6.59±0.01 (P<0.01) while EPA was without effect. The functional importance of V‐type H+ATPase‐activity in preserving pHihomeostasis at acidic extracellular oH levels was reflected by the impairment of O2−production at pHo6.70 when H+ATPase activity was inhibited: bafilomycin A1reduced O2−production from 13.9±1.0 to 9.3±0.6 nmoles/106cells/40 min, in control and bafilomycin A1‐treated cells, respectively (P≤0.05), while EPA had no effect. In subsequent studies, pHiwas independently manipulated using the ionophore nigericin. Lowering pHifrom 6.80 to 6.60 reduced O2−production from 15.3±1.8 to 9.8±1.6 nmoles/106cells/40 min (P≤0.05), indicating that the cytoplasmic acidification resulting from inhibition of H+ATPases at low pHocould account for the associated impairment of O2−production. In a more profoundly acidic environment (pHo6.35), H+ATPases remained active in regulating pHi, but could not preserve a sufficiently physiological pHito supprt respiratory burst activity. V‐type H+ATPases constitute the dominant mechanism by which the pHiof peritoneal Mψs is maintained in an acid

ISSN:0021-9541    

DOI:10.1002/jcp.1041570304

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

年代:1993

数据来源: WILEY

摘要:

AbstractColonies of small hepatocytes appeared after the culture of primary adult rat hepatocytes for 4 days in serum‐free Dulbecco's modified Eagle's medium containing 10 mM nicotinamide and 10 ng/ml of epidermal growth factor (EGF), acidic and basic fibroblast growth factors (FGF), hepatocyte growth factor (HGF), or transforming growth factor‐α (TGF‐α). Every colony consisted of cells that each had a single nucleus and a higher nucleus/cytoplasm ratio than surrounding hepatocytes, and immunocytochemically the cells induced by any mitogen were stained with albumin, transferrin, cytokeratin‐8 and ‐18. But these cells expressed neither cytokeratin‐7 nor‐19. When 6 × 105cells were plated on 35‐mm dishes, about 15 colonies per 1,000 attached cells were observed in the cultures treated with EGF, HGF, and TGF‐α. Although FGFs could also induce colonies, their number was less than half of the number induced by EGF. Furthermore, the numbers of colonies induced by the combinations of EGF + HGF, EGF + TGF‐α, and HGF + TGF‐α were not different from those of the colonies induced by each mitogen alone. To examine the ability of co‐mitogenic factors to induce small‐cell colonies, angiotensin‐II, insulin‐like growth factor‐I, norepinephrine, tumor necrosis factor, and vasopressin were used. In the cells cultured without EGF, these co‐mitogens neither stimulated DNA synthesis nor induced colonies. On the other hand, in cells cultured with both EGF and each co‐mitogen, although the DNA synthesis of the hepatocytes was enhanced, the number of colonies detected was not significantly different from the number which EGF alone could induce. These results showed that the small‐cell colonies in primary cultures of rat hepatocytes were inducible by EGF, HGF, TGF‐α, or FGFs and that the co‐mitogens did not influence the formation of

ISSN:0021-9541    

DOI:10.1002/jcp.1041570305

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

年代:1993

数据来源: WILEY

摘要:

AbstractLipid synthesis and secretion was measured in primary rat mammary epithelial cells cultured on basement matrix in medium supplemented with lactogenic hormones. The cells grew and differentiated to form alveolar‐like structures reminiscent of lactating mammary gland. They synthesized abundant triacylglycerol, containing fatty acids characteristic of rat milk (C10:O‐C14:0), using14C‐glucose,14C‐oleic acid or14C‐glycerol as precursors. Basal levels of triacylglycerol secretion were measured using14C‐oleic acid labeling; 1.3±0.3% of the labeled cellular triacylglycerol was secreted into the medium in 24 hours. Secreted lipid droplets were surrounded by a bilayer membrane with an electron‐dense inner coat characteristic of fat globules secreted by the mammary gland. The rate of triglycerol secretion was increased to 998±98% of control (P<0.01) by the addition of phorbol 12‐myristate 13‐acetate (PMA) in combination with staurosporine, a protein kinase inhibitcn. Several other protein kinase inhibitors, when combined with PMA, also markedly stimulated secretion. Effective protein kinase inhibitors included sphingosine (has diverse cellular effects including the inhibition of protein kinase C; 13‐fold increase in secretion), and KT5823 (a cGMP dependent protein kinase inhibitor; 5‐fold increase). KT5720 (a cAMP‐dependent protein kinase inhibitor) did not alter secretion. Kinase inhibitors were effective only in the presence of a phorbol ester. 4α‐phorbol‐12,13‐didecanoate, a phorbol ester which does not activate protein kinase C (PKC), could substitute for PMA. Lipid release was not mediated by disruption of cell‐cell tight junctions, as EGTA did not release lipid. Based on these observations we suggest that two signals are needed to enable or stimulate lipid secretion in cultured rat mammary epithelial cells: (1) inhibition of a protein kinase and (2) a PKC‐independent effect of phorbol ester. We have, for the first time, characterized a cell culture model suitable for studying lipid synthesis and secretion by mammary epit

ISSN:0021-9541    

DOI:10.1002/jcp.1041570306

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

年代:1993

数据来源: WILEY

摘要:

AbstractSix novel alkaloids that contain a fused tetracyclic pyrido[2,3,4‐kl]acridine ring system were purified recently from the Red Sea purple tunicate Eudistoma sp. Evaluation of the effects of these alkaloids on cultured neuroblastoma and fibroblast cells revealed that they possess potent growth regulatory properties, and affect cell shape and adhesion. In mouse neuroblastoma cells, the Eudistoma alkaloids inhibited cell proliferation and induced a process of differentiation during which the cels flattened onto the surface, increased considerably in size, and extended long neurites. In hamster fibroblasts the alkaloids slowed down cell multiplication, and caused an exceptional cell flattening or elongation. In a virustransformed derivative of the hamster fibroblasts the alkaloids restored many aspects of normal cell growth and morphology. In addition, several of the alkaloids mimicked the effects of cAMP analogs on two well‐characterized cAMP‐mediated processes involved in hepatic glucose metabolism–inhibition of pyruvate kinase (PK) activity and induction of mRNA for phosphoenolpyruvate carboxykinase (PEPCK). All these effects suggest that the Eudistoma alkaloids may act on the cAMP signaling system. However, a single application of these compounds was sufficient to completely block cell multiplication and to induce and sustain differentiation and “reverse transformation”. Furthermore, these effects were not readily reversible following removal of the drugs. In contrast, a single application of agents that mimic or elevate cAMP induced a transient response that waned with time in culture, and the effects induced by constant elevation of cAMP reverse rapidly following drug removal. We propose that theEudistomaalkaloids cause growth inhibition, differentiation, and reverse transformation by modifying the activity state of proteins that are involved in the regulation of cell shape and adhesion and serve as a target for the cAMP and/or other second messenger systems. © 1993 Wil

ISSN:0021-9541    

DOI:10.1002/jcp.1041570307

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

年代:1993

数据来源: WILEY

摘要:

AbstractThe study was conducted on human leukemia (K 562) cells to characterize the mechanisms implicated in the regulation of the polyamine spermicine (Spd) transport process. The antagonists of calmodulin, trifluoperazine (TFP), W‐7 (N‐[6‐aminohexyl]‐5‐chloro‐1‐naphthelenesulfonamide), or mellitin inhiblted significantly polyamine Spd uptake in these cells. The translocation of calmodulin towards plasma membrane and a concomitant decrease in its contents in cytosol were directly correlated with the time course increases similar to that of Spd uptake, indicating that calmodulin is recruited towards plasma membrane during the Spd transport process. Diminution of free intracellular calcium, (Ca2+)i, by preincubating the cells in BAPTA (bis[2‐amino‐5‐methylphenoxyl]‐ethane‐N,N′,N′,‐tetraacetate) buffer inhibited Spd transport significantly. Addition of lanthanum (LAN), a molecule known to inhibit Ca2efflux via Ca2+‐ATPase, curtailed Spd uptake by these cells. LAN inhibited Vmax, but not the Km, of Spd uptake, indicating that the former does not directly interact with the polyamine transporter; rather it regulates the transport process, probably via its action on Ca2+‐ATPase. Calmodulin‐stimulated uptake of45Ca2+by inside‐out vesicles of K 562 cells, a measure of Ca2+‐ATPase activity. Furthermore, addition of LAN inhibited both basal and calmodulin‐stimulated activity of Ca2+‐ATPase. Thapsigargin (THAP), a molecule known to elevate (Ca2+) i due to its action on the endoplasmic reticulum, increased Spd transport whereas addition of LAN inhibited THAP‐stimulated Spd transport activity. THAP increased free (Ca2+)i in these cells, and a pre‐addition of LAN to these cells curtailed the THAP‐stimulated increases of (Ca2+)i concentrations. Addition of Spd brought about elevations in (Ca2+)i contents. Caffeine also increased (Ca2+)i in these cells; however, it failed to stimulate significantly the Spd uptake process, indicating that (Ca2+)i which is involved in the regulation of polyamine transport pathways does not belong to the calcium‐induced calcium‐release (CICR) pool. Replacement of Ca2+from the incubation medium (i.e., 0% Ca2+) resulted in higher uptake activity as compared to that in 100% Ca2+medium, demonstrating that in 100% Ca2+medium the calcium efflux process is quickly compensated by calcium refilling/influx from the extracellular medium, while in 0% Ca2+medium there is perpetual efflux of (Ca2+)i which contributes to higher Spd uptake process. The results of this study suggest that an increase in free (Ca2+)i and its release from the cells via Ca2+ATPase, and concomitant activation of calmodulin, which controls Ca2+‐pump activity, are involved in the regulation of the Spd uptake

ISSN:0021-9541    

DOI:10.1002/jcp.1041570308

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

年代:1993

数据来源: WILEY

摘要:

AbstractAddition of ATP (>0.1 mM) to cultures of human breast cancer T47D cells resulted in an inhibition of cell proliferation. The inhibition was found to be specific for ATP, and dependent on its concentration. Growth inhibition continued for at least three days, although ATP and its hydrolysis products were metabolized within one day. Conditioned medium from ATP‐treated cultures (CM+) was found to inhibit the growth of cells that were not exposed to ATP. This is an indication that extracellular factors, besides ATP, are involved in the inhibition process. The inhibition was maintained after dialysis of the CM+, using an 8 kDa cut‐off membrane. Conditioned medium from untreated cultures (CM‐), however, only slightly affected cell growth. The data suggest that the CM+ ‐induced cell growth inhibition is mediated by an ATP‐activated growth inhibiting factor. Flow microfluorometry and thymidine incorporation experiments have shown that the growth arrest is mainly due to the elongation of the S‐phase of the cell cycle. © 1993 Wil

ISSN:0021-9541    

DOI:10.1002/jcp.1041570309

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

年代:1993

数据来源: WILEY

摘要:

AbstractThe initial events in glucose metabolism by all cells are the transport and phosphorylation of glucose. To quantify the relative contributions of these two processes to overall glucose utilization, we have developed an experimental approach for their in situ measurement as parallel processes. The method is based on the use of intracellular [2‐3H]glucose as a substrate for both the transporter and hexokinase, and involves simultaneous measurement of [2‐3H]glucose efflux and of3H2O released by phosphorylation. The Xenopus oocyte expression system was used to test the method, since in these cells transport and phosphory lation activities can be regulated by expression of mRNA or injection of foreign protein. Oocytes microinjected with [2‐3H]glucose showed no release of injected glucose, but did have saturable phosphorylation kinetics, with a Kmof 40 7μM and a Vmaxof 0.1 nmol/min/oocyte. Co‐injection of yeast hexokinase increased glucose phosphorylation by five‐fold. Expression of human glucose transporter (GLUT1) mRNA resulted in a 25‐30‐fold increase in the rate of saturable efflux of microinjected glucose compared to control oocytes. The kinetics of transport and phosphorylation of [2‐3H]glucose were analyzed by a multiple curve‐fitting program that provided estimates of kinetic coefficients for both processes from a single time course. The analysis showed that expression of GLUT1 shifted the rate‐limiting step in glucose utilization from transport to phosphorylation. A similar shift occurred at a three‐fold lower extracellular concentration of 2‐deoxyglucose. In a pancreatic beta cell line both transport and phosphorylation showed high Kmvalues, with phosphorylation as the limiting step. The in situ measurement of glucose transport and phosphorylation as parallel processes should be useful in defining the relative contributions of each step to overall glucose metabolism in other cell and tissue models

ISSN:0021-9541    

DOI:10.1002/jcp.1041570310

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

年代:1993

数据来源: WILEY

摘要:

AbstractRenal epitheliaL LLC‐PK1cell sheets incubated with tumor necrosis factor (TNF) undergo an acute, spontaneous, and rapidly reversible decrease in transepithelial resistance (TER). (Mullin et al., 1992). However, 24 to 72 h following TNF exposure, TER across the cell sheet increases 2‐fold. This later effect of TNF is also reversible, albeit slowly. The TER of TNF‐treated cell sheets then declines toward initia levels between 72 and 144 h following exposure to the cytokine. Whereas the long‐term increase in TER following TNF exposure is not associated with a decreased transepithelial14C‐mannitol flux (size selectivity), the charge (anionic) selectivity of the LLC‐PK1tight junction is decreased. Basal‐lateral (ouabain and bumetanide‐insensitive) Rb+and apical Na+‐dependent alpha‐methylglucoside (AMG) uptake into the cell are both reduced in cultures exposed to TNF 24 h earlier. Correspondingly, this long‐term effect on TER is accompanied by a 30% decrease in short circuit current(iSCC). Along with an observed increase in basallateral methylamino‐isobutyric acid (MeAIB) influx into the cells, an increased incorporation of [3H]‐thymidine into DNA indicates increased cell cycling after exposure to TNF. While the increase in cell cycling is not sustained for the duration of the elevation in TER, it does appear to initiate a sequence of events that lead to the sustained increase in TER. A decrease in the lateral intercellular space, observed between these epithelial cells after long‐term TNF exposure, may be a mechanism for the elevated TER following from the mitogenesis and/or transport changes. This overall long‐term tightening of an epithelium in response to TNF may function, in part, as a compensatory action of the epithelium to reestablish its effectiveness as a physiological barrier, following the acute effect o

ISSN:0021-9541    

DOI:10.1002/jcp.1041570311

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

年代:1993

数据来源: WILEY