摘要:

AbstractMixing feed fibroblasts with soluble collagen and serum‐supplemented culture medium at 37°C results in the entrapment of cells within the polymerizing collagen matrix. This cellular‐collagen complex is referred to as a fibroblast‐populated collagen lattice (FPCL). In time, this FPCL undergoes a reduction in size called lattice contraction. The proposed mechanism for lattice contraction is cellular force produced by cytoplasmic microfilaments which organize collagen fibrils compacting the matrix. When the regulatory subunits of myosin, myosin light chains, are phosphorylated by myosin light chain kinase (MLCK), myosin ATPase activity is increased and actin‐myosin dynamic filament sliding occurs. Elevated levels of myosin ATPase are required for maximal lattice contraction. Cholera toxin inhibits lattice contraction by increasing intracellular levels of cAMP. It is proposed that increased cytoplasmic concentrations of cAMP promote phosphorylation of MLCK, the enzyme important for maximizing myosin ATPase activity. Phosphorylating MLCK in vitro inhibits activity by decreasing its sensitivity to calcium‐calmodulin complex. A decrease in MLCK activity would result in lower levels of myosin ATPase activity. MLCK, purified from turkey gizzard, was subjected to limited proteolytic digestion to produce calmodulin‐independent‐MLCK. The partially digested kinase does not require calcium‐calmodulin for activation. Independent‐MLCK is not subject to inhibition by phosphorylation. The electroporetic inoculation of independent‐MLCK into fibroblasts before FPCL manufacture produced enhanced lattice contraction. Lattice contraction, in the presence of cholera toxin, was restored to normal levels by the prior electroporetic introduction of independent‐MLCK. These findings support the hypothesis that increases in cAMP hinder lattice contraction by a mechanism involving inhibition of

ISSN:0021-9541    

DOI:10.1002/jcp.1041460102

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

年代:1991

数据来源: WILEY

摘要:

AbstractIn bovine aortic or capillary endothelial cells (ECs) incubated under hypoxic conditions, cell growth was slowed in a dose‐dependent manner at lower oxygen concentrations, as progression into S phase from G1 was inhibited, concomitant with decreased thymidine kinase activity. Monolayers grown to confluence in ambient air, wounded, and then transferred to hypoxia showed decreased ability to repair the wound, as a result of both decreased motility and cell division. Hypoxic ECs demonstrated a ≈3‐fold increase in the total number of high‐affinity fibroblast growth factor receptors, and levels of endogenous FGF were suppressed. Consistent with the presence of functional FGF receptors, addition of basic FGF overcame, at least in part, hypoxia‐mediated suppression of EC growth, and enhanced wound repair in hypoxia, stimulating both motility and cell division. Despite slower growth in hypoxia, ECs could achieve confluence, and the monolayers consisted of larger cells with altered assembly of the actin‐based cytoskeleton and small gaps between contiguous cells. The permeability of these hypoxic EC monolayers to macromolecules and lower molecular weight solutes was increased. Cell surface coagulant properties were also perturbed: the anticoagulant cofactor thrombomodulin was suppressed, and a novel Factor X activator appeared on the EC surface. These data indicate that micro‐ and macrovascular ECs can grow and be maintained at low oxygen tensions, but hypoxic endothelium exhibits a range of altered functional properties which can potentially contribute to the pathogenesis of vas

ISSN:0021-9541    

DOI:10.1002/jcp.1041460103

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

年代:1991

数据来源: WILEY

摘要:

AbstractRat hepatoma cells (Fu5AH) were studied as a model for the net delivery of apoE‐free high‐density lipoprotein (HDL) cholesterol to a cell. Incubating cells with HDL results in (1) a decrease in both media‐free cholesterol and cholesteryl ester concentration; (2) decreased cell sterol synthesis; and (3) increased cell cholesteryl ester synthesis. HDL cholesteryl ester uptake is increased when cells are incubated for 18 hr in cholesterol poor media. Coincubation of3H‐cholesteryl ester‐labeled low‐density lipoprotein (LDL) with 50 μM chloroquine or 25 μM monensin results in a decrease in the cellular free cholesterol/cholesteryl ester (FC/CE) isotope ratio, indicating an inhibition in the conversion of cholesteryl ester to free cholesterol. In contrast, chloroquine and monensin do not alter the cellular FC/CE isotope ratio for3H‐CE HDL. This evidence indicates that acidic lysosomal cholesteryl ester hydrolase does not account for the hydrolysis of HDL‐CE. Free cholesterol generated from3H‐cholesteryl ester of both LDL and HDL is reesterified intracellularly. At higher HDL concentrations (above 50 μg/ml) HDL cholesteryl ester hydrolysis is sensitive to chloroquine. We propose that an extralysosomal pathway is operating in the metabolism of HDL cholesterol and that at higher HDL concentrations a lysosomal pathway may be functioning in addition to an ex

ISSN:0021-9541    

DOI:10.1002/jcp.1041460104

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

年代:1991

数据来源: WILEY

摘要:

AbstractTo elucidate mechanisms of mercury toxicity, the cell membrane potential has been determined continuously in cultured kidney (MDCK)‐cells during reversible application of mercury ions to extracellular perfusate. Exposure of the cells to 1μM mercury ions is followed by rapid, sustained, and slowly reversible hyperpolarization of the cell membrane, increase of cell membrane potassium selectivity, and decrease of cell membrane resistance. Thus, mercury ions enhance the potassium conductance of the cell membrane. Half maximal hyperpolarizing effect is elicited by ≈0.2 μM. Higher concentrations of mercury ions (>10 μM) eventually depolarize the cell membrane. At extracellular calcium activity reduced to less than 0.1 μM, 1 μM mercury ions still leads to a sustained hyperpolarization and increase of potassium selectivity of the cell membrane. As evident from fluorescence measurements, 10 μM, but not 1 μM mercury ions leads to a rapid increase of intracellular calcium activity. Pretreatment of the cells with either pertussis toxin or cholera toxin does not blunt the hyperpolarizing effect of mercury ions. In conclusion, mercury ions activate the potassium conductance by a mechanism independent of increase of intracellular calcium activity and of cholera toxin‐or pertussis toxin‐sensitive G‐proteins. This activation of potassium conductance may account for early effects of mercury intoxication, su

ISSN:0021-9541    

DOI:10.1002/jcp.1041460105

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

年代:1991

数据来源: WILEY

摘要:

AbstractIncubation of cultured cells in hypertonic medium and sodium‐free medium have been shown to block transport at two different stages along the endocytic pathway. To determine the effects of these treatments on the exocytic pathway, we studied the transport of the membrane glycoprotein of vesicular stomatitis virus (VSV‐G) in cells infected with tsO45 mutant virus. This mutant synthesizes a VSV‐G that accumulates in the endoplasmic reticulum (ER) when cells are incubated at 39.5°C. In addition, VSV‐G accumulates in the post‐ER pre‐Golgi compartment when cells are incubated at 15°C and in the trans‐Golgi network (TGN) when cells are incubated at 18°C. Upon transfer of cells to 32°C in control medium, VSV‐G exits each of these compartments and is transported to the cell surface. Incubation in sodium‐free medium at 32°C did not block transport from any of these three compartments. In contrast, incubation in hypertonic medium blocked export from the ER, transport from the pre‐Golgi compartment to the Golgi complex, and transport from the TGN to the cell surface. Our results, in combination with previous studies, suggest that hypertonic medium blocks at least five distinct transport steps: the three exocytic steps described here, endocytosis from the cell surface, and transport of cell surface proteins into the Golgi complex. This raises the possibility that vesicular transport in different parts of the cell shares common elements that are inh

ISSN:0021-9541    

DOI:10.1002/jcp.1041460106

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

年代:1991

数据来源: WILEY

摘要:

AbstractAtrial natriuretic peptide (ANP) binding and ANP‐induced increases in cyclic guanosine monophosphate (cGMP) levels have been observed in brain microvessels (Chabrier et al., 1987; Steardo and Nathanson, 1987), suggesting that this fluid‐regulating hormone may play a role in the fluid homeostasis of the brain. This study was initiated to characterize the ANP receptors in primary cultures of brain microvessel endothelial cells (BMECs). The apparent equilibrium dissociation constant, Kd, for ANP increased from 0.25 nM to 2.5 nM, and the number of ANP binding sites as determined by Scatchard analysis increased from 7,100 to 170,000 sites/cell between 2 and 10 days of culture following monolayer formation. Time‐ and concentration‐dependent studies on the stimulation of cGMP levels by ANP indicated that guanylate cyclase‐linked ANP receptors were present in BMECs. The relative abilities of ANP, brain natriuretic peptide (BNP), and a truncated analog of ANP containing amino acids 5–27 (ANP 5–27) to modulate the accumulation of cGMP was found to be ANP>BNP ≫ ANP 5–27. Affinity cross‐linking with disuccinimidyl suberate and radiolabeled ANP followed by gel electrophoresis under reducing conditions demonstrated a single band corresponding to the 60–70 kD receptor, indicating the presence of the nonguanylate cyclase‐linked ANP receptor. Radiolabeled ANP binding was examined in the presence of various concentrations of either ANP, BNP, or ANP 5–27 and suggested that a large proportion of the ANP receptors present in blood‐brain barrier endothelial cells bind all of these ligands similarly. These data indicate both guanylate cyclase linked and nonguanylate cyclase linked receptors are present on BMECs and that a higher proportion of the nonguanylate cyclase linked receptors is expressed. This in vitro culture system may provide a valuable tool for the examination of ANP receptor expression and function in blood‐br

ISSN:0021-9541    

DOI:10.1002/jcp.1041460107

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

年代:1991

数据来源: WILEY

摘要:

AbstractDetermination of the transient increase in plasma homocysteine following administration of excess methionine is an established procedure for the diagnosis of defects in homocysteine metabolism in patients. This so‐called methionine loading test has been used for 25 years, but the knowledge of the response of various cell types to excess methionine is limited. In the present paper we investigated homocysteine export from various cell types cultured in the presence of increasing concentrations (15–1,000 μM) of methionine. For comparison of homocysteine export, the export rates per million cells were plotted versus cell density for proliferating cells, and versus time for quiescent cells. The homocysteine export from growing cells was greatest during early to mid‐exponential growth phase, and then decreased as a function of cell density. The export rate was higher from phytohemagglutinin‐stimulated than non‐stimulated lymphocytes, and higher from proliferating than from quiescent fibroblasts. The hepatocytes showed highest export rate among the cell types investigated. The enhancement of homocysteine export by excess methionine ranged from no stimulation to marked enhancement, depending on cell type investigated, and three different response patterns could be distinguished: (1) quiescent fibroblasts and growing murine lymphoma cell showed no significant increase in homocysteine export following methionine loading; export from human lymphocytes was only slightly enhanced in the presence of excess methionine; (2) the homocysteine export from proliferating hepatoma cells and benign and transformed fibroblasts was stimulated three to eightfold by increasing the methionine concentration in the medium from 15 to 1,000 μM; and (3) the response to methionine loading was particularly increased (about 15‐fold) in non‐transformed primary hepatocytes in stationary culture. The results outline a potentially useful procedure for the comparison of homocysteine export during cell growth in the presence of various concentrations of methionine. The results are discussed in relation to the special feature of homocysteine metabolism in various cell types and tissues including liver, and to the possible source of plasma homocysteine following methionine

ISSN:0021-9541    

DOI:10.1002/jcp.1041460108

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

年代:1991

数据来源: WILEY

摘要:

AbstractThe epidermal growth factor (EGF) receptor interacts with structural elements of A431 cells and remains associated with the cytoskeleton following extraction with nonionic detergents. Extraction of cells with 0.15% Triton X‐100 resulted in detection of only approximately 40% of the EGF binding sites on the cytoskeleton. If the cells were exposed to EGF prior to extraction, approximately twofold higher levels of low‐affinity EGF binding sites were detected. The difference in number of EGF binding sites was not a consequence of differences in numbers of EGF receptors associated with the cytoskeleton; equal amounts of35S‐labeled receptor were immunoprecipitated from the cytoskeletons of both control and EGF‐treated cells. The effect of EGF pretreatment on binding activity was coincident with a change in the mobility of the receptor from a doublet of Mr∼ 160–180 kDa to a single sharp band at 180 kDa. The alteration in receptor mobility was not a simple consequence of receptor phosphorylation in that the alteration was not reversed by alkaline phosphatase treatment, nor was the shift produced by treatment of the cells with phorbol ester. The two EGF receptor species demonstrated differential susceptibility to V8 proteinase digestion. The EGF‐induced 180 kDa species was preferentially digested by the proteinase relative to the 160 kDa species, indicating that EGF binding results in a conformational change in the receptor. The EGF‐mediated preservation of binding activity and altered conformation may be related to receptor

ISSN:0021-9541    

DOI:10.1002/jcp.1041460109

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

年代:1991

数据来源: WILEY

摘要:

AbstractProgrammed death (apoptosis) of the rat myelocytic leukemic cell line IPC‐81 was triggered by cyclic adenosine monophosphate (cAMP) analogs or by agents (cholera toxin, prostaglandins) increasing the endogenous cAMP level. The induction of cell death by cholera toxin was preceded by increased activation of cAMP‐kinase. Cell lysis started already 5 hr after cAMP challenge and was preceded by internucleosomal DNA fragmentation and morphological changes characteristic of apoptosis. The cell suicide could be prevented by inhibitors of macromolecular synthesis. cAMP analogs induced cell death in a positively cooperative manner (apparent Hill coefficient of 2.9), indicating that triggering of the apoptotic process was under stringent control. There was a strong synergism between cAMP analogs complementing each other in the activation of cAMP‐dependent protein kinase I (cAKI). No such synergism was noted for analogs complementing each other in the activation of cAKII. It is concluded that apoptosis can be induced solely by activation of cAKI. The IPC‐81 cells expressed about four times more cAKI than cAKII. The expression of cAK subunits, on the protein and mRNA levels, was only minimally affected by cholera toxin tr

ISSN:0021-9541    

DOI:10.1002/jcp.1041460110

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

年代:1991

数据来源: WILEY

摘要:

AbstractRecent reports suggest that prostaglandins, rather than cAMP, play a major role in mediating cholera toxin‐induced water and electrolyte secretion from rabbit intestinal loops. We examined the role of prostaglandins in mediating toxin‐induced pancreatic and gastric exocrine secretion. In these tissues, indomethacin, a potent inhibitor of prostaglandin synthesis, did not alter the stimulatory effects of cholera toxin on increases in cellular cAMP or enzyme secretion. Moreover, the addition of cholera toxin did not alter prostaglandin E2release from either tissue. In contrast to their effects in rabbit intestinal loops, prostaglandins do not regulate cholera toxin‐induced enzyme secretion from the guinea pig pancreas or st

ISSN:0021-9541    

DOI:10.1002/jcp.1041460111

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

年代:1991

数据来源: WILEY