摘要:

Abstract5‐hydroxytryptamine (5‐HT) is a mitogen for fibroblasts, vascular smooth muscle cells, renal mesangial cells, and jejunal crypt cells. The human carcinoid cell line (termed BON) that we established in our laboratory from a pancreatic carcinoid tumor produces and secretes 5‐HT. In this study, therefore, we examined the effect of 5‐HT on growth of BON cells. Furthermore, by use of selective 5‐HT receptor antagonists, we examined receptor and post‐receptor mechanisms by which 5‐HT‐induced responses were produced. 5‐HT stimulated growth of BON cells. 5‐HT stimulated phosphatidylinositol (PI) hydrolysis in a dose‐dependent fashion and inhibited cyclic AMP production in a dose‐dependent fashion. The 5‐HT1A/1Breceptor antagonist, SDZ 21–009, prevented the reduction of cyclic AMP production evoked by 5‐HT and inhibited the mitogenic action of 5‐HT. The 5‐HT1C/2receptor antagonist, mesulergine, competitively inhibited PI hydrolysis, but did not affect the mitogenic action of 5‐HT. The mitogenic action of 5‐HT and the reduction of cyclic AMP production evoked by 5‐HT were also inhibited by pertussis toxin. These results suggest that 5‐HT is an autocrine growth factor for BON cells and that mitogenic mechanism of 5‐HT involves receptor‐mediated toxin‐sensitive GTP binding protein. 8‐bromo‐cyclic AMP inhibited growth of BON cells whereas 8‐bromo‐cyclic GMP had no effect on cell growth. Involvement of protein kinase A in BON cell growth regulation was confirmed by the observation that a cAMP‐dependent protein kin

ISSN:0021-9541    

DOI:10.1002/jcp.1041500102

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

年代:1992

数据来源: WILEY

摘要:

AbstractIn investigating the role of cell‐extracellular matrix interactions in cell adhesion and growth control, the effects of heparin on cell‐collagen interactions were examined. Exponentially growing Balb/c‐3T3 fibroblasts were radiolabelled with3H‐thymidine and detached from tissue culture surfaces using EDTA, and cell attachment to various types of collagen substrata was assayed in the presence or absence of heparin or other glycosaminoglycans (GAGs) or dextran sulfate (40 K). Cells attached readily (70–90%) to films of types I and V, but not to type III collagen. The number of cells bound to types I and V collagen films was inhibited by 10–50% when heparin was present from 0.1–100 μg/ml. Cell‐collagen attachment was also inhibited by dextran sulfate, and to a lesser extent by dermatan sulfate, but chondroitin sulfates A and C and hyaluronic acid showed no effect. Heparin was active even at early time points in the adhesion assay, suggesting it may disrupt cell‐collagen attachment. To study the effects of heparin in modulating cell growth on collagen, growth arrested cells cultured on type I collagen films were serum stimulated in the presence of heparin or other GAGs for 3 days. Growth was inhibited (>40%) only by heparin and dextran sulfate. Interaction of heparin fragments (Mr≤ 6KD) with type I collagen was analyzed by affinity co‐electrophoresis (Lee and Lander, 1991) and showed higher affinity heparin binding to native as compared with denatured collagen. These data suggest that sites within native collagen may mediate Balb cell–collagen and heparin‐collagen interactions, and such interactions may be relevant towards understanding heparin's antiproliferative act

ISSN:0021-9541    

DOI:10.1002/jcp.1041500103

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

年代:1992

数据来源: WILEY

摘要:

AbstractKaryotypic and phenotypic changes were found in human adult endothelial cells (EC) during aging in vitro. A trisomy of chromosome 11 was found in 11 out of 12 EC cultures examined, derived from 9 cell lines from 8 donors. The incidence of this trisomy in some cell lines increased over time from 0% to as much as 100% near the end of their in vitro life span. A number of oncogenes and other important genes are on chromosome 11. These genes might play a role in the changes observed. An increase in the percentage of polyploid cells was also found near the end of the in vitro life span in 6 lines. The cellular levels of two gene products characteristic of the EC, von Willebrand factor (vWF) or Factor VIII, and angiotensin converting enzyme (ACE) were also monitored. vWf was studied in 2 lines and was decreased in both with serial passage. ACE decreased in three out of the four lines examined. These chromosomal and phenotypic changes which occur with increasing age in vitro make the endothelial cell a suitable model to study in vitro culture‐related changes, senescence, cardiovascular disease, and tumorigenesi

ISSN:0021-9541    

DOI:10.1002/jcp.1041500104

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

年代:1992

数据来源: WILEY

摘要:

AbstractA peripheral nervous system cell line RT4‐B, established by lmada and Sueoka (Dev. Biol., 66:97–108, 1978), was shown to respond to serotonin [5‐hydroxy‐tryptamine (5‐HT)] and catecholamines. 5‐HT induced a small and transient increase in cytosolic free Ca2+concentration ([Ca2+]i) in the RT4‐B cells. The increase was effectively blocked by 5‐HT2receptor antagonists (spiperone, ritanserin and mianserin), but not by a 5‐HT3receptor antagonist (MDL72222), or a α1‐adrenergic receptor antagonist (prazosin), indicating that RT4‐B cells express 5‐HT2receptors. On the other hand, catecholamines increased cyclic AMP production by RT4‐B. The order of potency for stimulating cyclic AMP synthesis was isoproterenol>epinephrine ≫ norepinephrine ≫ dopamine, and the stimulation was effectively inhibited by the nonselective β‐adrenergic receptor antagonist propranolol, but not by the β1‐adrenergic receptor antagonist atenolol, suggesting that RT4‐B cells express β2‐adrenergic receptors.The differentiating agent N6,2′‐O‐dibutyryladenosine 3′,5′‐monophosphate (dibutyryl‐cAMP) enhanced the 5‐HT‐induced [Ca2+]iincrease, but not the catecholamine‐induced cyclic AMP production. The increase in the 5‐HT response paralleled the increase in the density of 5‐HT2receptors. n‐Butyric acid (2 mM) and 8‐bromoadenosine 3′,5′‐monophosphate (1 mM) also increased the 5‐HT response, and the sum of these increases was nearly equal to that induced by dibutyryl‐cAMP.These results indicate that RT4‐B is a novel model cell line for the study of 5‐HT2and β2‐adrenergic receptors and their second messenger responses

ISSN:0021-9541    

DOI:10.1002/jcp.1041500105

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

年代:1992

数据来源: WILEY

摘要:

AbstractPrevious results (Fressinaud, C., Sarliève, L.L., and Labourdette, G. J. J. Cell. Physiol.,141:667–674, 1989b) have shown that cerebroside sulfotransferase (CST; EC 2.8.2.11) is enriched in pure rat oligodendrocyte (OL) cultures and that its activity is increased by factors mitogenic for OL precursors and galactocere‐broside (GC) expressing OL, such as basic fibroblast growth factor (bFGF), platelet‐derived growth factor, and high insulin concentrations. In contrast, transforming growth factor β or low insulin concentrations were found to be ineffective in this culture system. As bFGF mainly enhanced the proliferation of OL precursors (GC negative cells) rather than that of differentiated (GC +) cells, a relationship between OL precursor proliferation and CST increase was suggested. This hypothesis was first tested in 20‐day‐old OL cultures grown in chemically defined medium. The dose‐response curve of [125I] lododeoxyuridine ([125I]dUrd) incorporation toward bFGF was parallel to that of CST specific activity, and maximal stimulation was reached at 5 ng/ml bFGF for both. In contrast, 2′,3′‐cyclic nucleotide 3′‐phosphodiesterase (CNP; EC 3.1.4.37) specific activity decreased after bFGF treatment. To determine if CST increase was linked to the proliferation of OL precursors induced by bFGF, cell proliferation was blocked by cytosine arabinoside (ARA‐C). From 10−8to 10−5M ARA‐C there was a dose‐dependent inhibition of cell proliferation and a decrease in CST specific activity, whereas CNP specific activity was enhanced. When the cells were treated with bFGF and 10−6M ARA‐C together, the proliferation was completely blocked and CST activity decreased by 72% below control values, whereas CNP activity was not significantly decreased. Immunocytochemical studies showed that the number of sulfatide‐expressing cells and the number of cycling cells were increased after bFGF treatment, but that there was no overlapping between these two populations. Taken together these results suggest that CST activity and sulfatide expression appear shortly af

ISSN:0021-9541    

DOI:10.1002/jcp.1041500106

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

年代:1992

数据来源: WILEY

摘要:

AbstractSensitivity of cultured chick myotubes to alkaline earth metal ions was investigated by recording contractile isometric tension through a semiconductor transducer. The myotubes were obtained by culturing myoblasts of chick embryo breast muscles, and skinned chemically before physiological experiments. Contractions developed in response to Ca2+in a bathing medium higher than 3 × 10−7M and reached maximum at 1 × 10−5M. Sr2+was less effective than Ca2+; the threshold concentration was 1 × 10−5M and the tension reached maximum at 1 × 10−3M. Ba2+was the least effective among the three alkaline earth metal ions; only one fifth of the Ca2+‐induced maximum tension was attained at 1 × 10−3M. The sensitivity was similar to that of the mature pectoral muscle fiber, a fast twitch muscle fiber, rather than that of the anterior latissimus dorsi, a slow tonic muscle fiber. The sensitivity was shown to be dependent on its troponin C by replacing it with troponin C from the mature pectoral or cardiac muscle. This indicates that TnC of a fast‐muscle type is expressed in the cultured chick myotube as in the mature pectoral muscle. The contractile apparatus was thus shown to be well developed in the cultured myotube with characteristics similar to the mature fast t

ISSN:0021-9541    

DOI:10.1002/jcp.1041500107

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

年代:1992

数据来源: WILEY

摘要:

AbstractIn vitro tissue culture techniques were employed to study the effects of bacterial endotoxins on the growth of normal epithelial cells from the human ureter (NHU). Primary cultures of NHU cells were initiated from explant outgrowth cultures of human ureteral tissue and cultured on collagen gel in F‐12* medium containing 1% fetal calf serum (FCS). Optimal clonal growth of secondary cultures of NHU cells seeded at relatively low seeding cell densities, directly on plastic dishes, was achieved in F‐12* medium containing bovine pituitary extract (0.5% BPE) and 0.05% BSA. Results indicated that insulin in the F‐12* medium could be replaced by three orders of magnitude less IGF‐1. Further clonal growth experiments demonstrated that PGE1is growth stimulatory and can replace BPE as a growth factor requirement. This finding was in agreement with the fact that BPE growth requirement could be replaced by cholera toxin or dibutyryl cAMP. These results suggested that both BPE and cholera toxin operated by activation of a cAMP‐dependent mitogenic pathway. Seven gram‐negative bacterial lipopolysaccharides (LPS) and three gram‐positive bacterial lipotechoic acids (LT) were tested for their effects on NHU clonal growth. Three out of the five LPS derived fromEscherichia coli(strains 055:B5, 0128:B12, and 0127:B8), LPS fromKlebsiella pneumoniae, and LPS fromPseudomonas aeruginosaall showed significant growth inhibitory effects at minimally effective doses ranging from 5 to 25 μg/ml. LPS derived fromE. colistrain (0111:B4) had no growth effects at the highest concentration tested (100 μg/ml). In contrast, LT derived fromStreptococcus pyogenes, S. faecalis, Staphylococcus aureas, andBacillus subtilisall markedly enhanced clonal growth at concentrations ranging from 1 μg/ml<[LT]<50 μg/ml. LT fromStrep. pyogeneswas inhibitory to clonal growth at 100 μg/ml. The growth inhibitory effects of LPS were shown to be sensitive to the presence of hydrocortisone in the growth medium, indicating that LPS effects on growth are mediated via the arachidonic acid cascade. We speculate that these results indicate a link between the susceptibility of uroepithelial tissue to the pathogenic microflora seen in urinary tract diseases and the differential sensitivity of proliferation‐competent uroepithelial cells to growth inhibition by LPS produced by gram‐negative bacteria. However, further studies with uropathogenic serotypes will be necessary to corroborate this possibility. The growth‐stimulating activity of LTs produced by gram‐positive bacteria may be due to their ability to bind to cell‐associated fibronectin and to activate the fibronectin receptor as part of ligand receptor–induced mitogenic tr

ISSN:0021-9541    

DOI:10.1002/jcp.1041500108

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

年代:1992

数据来源: WILEY

摘要:

AbstractIL‐1 inhibits erythropoiesis in vivo and in vitro. This inhibition was studied by comparing the effect of recombinant human IL‐1 (rhIL‐1) on highly purified CFU‐erythroid (E) generated from peripheral blood burst‐forming units‐erythroid (BFU‐E) (mean purity 44.4%) with its effect on unpurified marrow CFU‐E (mean purity 0.36%). Colony formation by marrow CFU‐E was significantly inhibited by rhIL‐1, while colony formation by highly purified CFU‐E was not inhibited. However, purified CFU‐E colonies were inhibited by rhIL‐1 in the presence of autologous T‐lymphocytes, and also by cell‐free conditioned medium prepared from T‐lymphocytes stimulated by rhIL‐1. This inhibitory effect was ablated by neutralizing antibodies to γinterferon (IFN), but not by antibodies to human IL‐1, tumor necrosis factor, or βIFN. Colony formation by highly purified CFU‐E was also inhibited by recombinant human γIFN (rhγIFN). IL‐1 and γIFN play significant roles in the pathogenesis of the anemia of chronic disease. These studies indicate that rhIL‐1 inhibits CFU‐E colony formation by an indirect mechanism involving T‐lymphocytes and requiring γIFN and that γIFN itself

ISSN:0021-9541    

DOI:10.1002/jcp.1041500109

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

年代:1992

数据来源: WILEY

摘要:

AbstractThrombin, a potent platelet activating agent, has previously been found to increase intracellular calcium levels and/or thromboxane A2synthesis in leukemic cell lines exhibiting specific markers of the megakaryocyte/platelet lineage. However, its functional role on these cells has not been defined. As thrombin is implicated in the regulation of cellular proliferation or differentiation in various other cell types, we investigated the functional effects of thrombin on the megakaryoblastic MEG‐01 cell line, and further explored its receptor coupling mechanisms on these cells. We observed that thrombin caused in 1% serum containing culture medium, a reduction in the proliferation of MEG‐01 cells, without affecting their differentiation stage as determined by the expression of platelet glycoproteins GPIIb/IIIa and GPIb, FVIII‐related‐antigen and cell‐size measurement, which are specific markers for megakaryocyte maturation. In increases in cAMP levels, and in inositol‐trisphosphate formation and intracellular Ca2+levels. All these responses required thrombin proteolytic activity. The lipoxygenase inhibitor, nordihydroguaiaretic acid, blunted thrombin‐induced calcium increase without affecting thrombin‐induced increase in cAMP levels, suggesting different thrombin coupling mechanisms with these two second messenger pathways. In addition, the inhibitory effect of thrombin on MEG‐01 cell growth was mimicked by cAMP level enhancing agents such as forskolin, prostaglandin E1and Bt2cAMP. These results suggest the involvement of a cAMP‐dependent mechanism in the thrombin‐induced reduction

ISSN:0021-9541    

DOI:10.1002/jcp.1041500110

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

年代:1992

数据来源: WILEY

摘要:

AbstractCultured monkey retinal pigment epithelial (RPE) cells rapidly secrete large amounts of insulin‐like growth factor binding proteins (IGF‐BPs). IGF‐II tracer binding activity in conditioned media is two to three times greater than that of IGF‐I. Under reducing SDS‐PAGE conditions,125I‐IGF affinity‐crosslinked binding protein (BP) is visualized as a broad band between 36 ± 2.9 and 49 ± 3.3 kDa. Because the electrophoretic mobility of the crosslinked BP is increased under non‐reducing conditions (33–45 kDa), intramolecular sulfhydryl bonding may be present. Frequently, the radiographic band representing affinity‐crosslinked binding protein exhibits a complex pattern of non‐uniform densities that suggests structural or functional IGF‐BP micro‐heterogeneity. IGF‐BPs synthesized by RPE also exhibit heterogeneity with respect to the absence or presence of oligosaccharide side chains. In particular, the larger, but not the mid‐sized or smaller IGF‐BPs exhibit side chains linked to the core protein with N‐glycosidic linkage. None of the crosslinked IGF‐BPs exhibit O‐linked side chains. Long‐term (12, 24, 48 hr) conditioning studies revealed that IGF‐BP fails to accumulate in culture media beyond 12 hr, but that replacement of conditioned media with fresh media allows a second period of binding protein accumulation. Other short‐term (12 hr) experiments indicate that, in fresh medium, the levels of IGF‐BP increase during the first 6–8 hr and then remain stable. To examine the processes contributing to these steady state levels of IGF‐BP, aliquots of 8‐hr conditioned medium were removed from the cells and either frozen on dry ice or incubated at 37°C for 16 hr. Importantly, it was found that incubation at 37°C resulted in a near total loss of binding activity. This is the first report of IGF‐BP degrading activity in a cell culture system. These findings indicate that (1) primate RPE cells rapidly secrete a complex mixture of N‐glycosylated and non‐glycosylated IGF‐BPs, and (2) the steady state levels of secreted IGF‐BP are tightly regulated at least in part through a concomitant IGF‐BP inactivating activity. Cultured RPE cells may be of utility in examining the mechanisms of

ISSN:0021-9541    

DOI:10.1002/jcp.1041500111

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

年代:1992

数据来源: WILEY