bluuebunny/crossref_metadata_2025 huggingface dataset:
Serotonin produces a configurational change of cultured smooth muscle cells that is associated with elevation of intracellular cAMP
Authors: Lee Sheu‐Ling , Fanburg Barry L.
Published: February 2005

Early passaged bovine pulmonary artery smooth muscle cells (SMC) respond to serotonin (5‐HT) by developing a reversible change in configuration. (Lee et al. J. Cell. Physiol. 138:145, 1989). This configurational change does not occur in pulmonary artery endothelial cells (EC) subjected to 5‐HT and is adenosine triphosphate (ATP) dependent, lost with passage of SMC, and inhibited by various agents that block high‐affinity 5‐HT uptake. We now report a second configurational change (also dendritic formation) of SMC produced by 5‐HT only in the presence of isobutylmethylxanthine (IBMX), an inhibitor of phosphodiesterase. This configurational change was also ATP dependent, but unlike the first response, (Lee et al., 1989), it occurred in both first and later passaged SMC and was not inhibited by blockade of 5‐HT uptake. Also, unlike the response with 5‐HT alone that failed to elevate cAMP, this one was associated with a large elevation of cAMP (eight fold above control values), similar to the response to the β‐agonist isoproterenol, plus IBMX. The second response was not blocked by a variety of 5‐HT receptor antagonists but was reproduced by (±)‐8‐hydroxy‐DPAT HBr (8‐OH‐DPAT), a reputed 5‐HT1A agonist. The response was not dependent upon Ca2+ and was blocked by 1–2 mM n‐phenylanthranilic acid or anthracene‐9‐carboxylic acid, electrically conductive Cl− channel inhibitors. Hence, 5‐HT in the presence of IBMX causes a marked elevation of cAMP of SMC and this elevation in cAMP likely results in a cellular configurational change through a Cl− channel‐dependent mechanism similar to that we previously described for EC in the presence of β‐adrenergic agonist stimulation (Ueda et al. Circ. Res. 66:951, 1990). EC do not show a similar response to 5‐HT possibly because cAMP is not adequately elevated, even in the presence of IBMX, to enhance Cl− channel activity. We propose that our observations indicate the presence of two sites of action of 5‐HT on the smooth muscle cell, one intracellularly and another at a cell surface receptor.
Lipopolysaccharides decrease angiotensin converting enzyme activity expressed by cultured human endothelial cells
Authors: Watanabe Kentaro , Lam George , Keresztes Roger S. , Jaffe Eric A.
Published: February 2005

Angiotensin converting enzyme (ACE) is present on endothelial cells and plays a role in regulating blood pressure in vivo by converting angiotensin I to angiotensin II and metabolizing bradykinin. Since ACE activity is decreased in vivo in sepsis, the ability of lipopolysaccharide (LPS) to suppress endothelial cell ACE activity was tested by culturing human umbilical vein endothelial cells (HUVEC) for 0–72 hr with or without LPS and then measuring ACE activity. ACE activity in intact HUVEC monolayers incubated with LPS (10 μg/ml) decreased markedly with time and was inhibited by 33%, 71%, and 76% after 24 hr, 48 hr, and 72 hr, respectively, when compared with control, untreated cells. The inhibitory effect of LPS was partially reversible upon removal of the LPS and further incubation in the absence of LPS. The LPS‐induced decrease in ACE activity was dependent on the concentrations of LPS (IC50 = 15 ng/ml at 24 hr) and was detectable at LPS concentrations as low as 1 ng/ml. That LPS decreased the Vmax of ACE in the absence of cytotoxicity and without a change in Km suggests that LPS decreased the amount of ACE present on the HUVEC cell membrane. While some LPS serotypes (Escherichia coli 0111 :B4 and 055 :B5, S. minnesota) were more potent inhibitors of ACE activity than others (E. coli 026:B6 and S. marcescens), all LPS serotypes tested were inhibitory. These finding suggest that LPS decreases endothelial ACE activity in septic patients; in turn, this decrease in ACE activity may decrease angiotensin II production and bradykinin catabolism and thus play a role in the pathogenesis of septic shock.
Decreased response to epidermal growth factor during cellular senescence in cultured human microvascular endothelial cells
Authors: Matsuda Takao , Okamura Kazuki , Sato Yasufumi , Morimoto Akio , Ono Mayumi , Kohno Kimitoshi , Kuwano Michihiko
Published: February 2005

We have previously demonstrated that epidermal growth factor (EGF) induces cell migration, tissue‐type plasminogen activator synthesis, as well as tubular formation in microvascular endothelial cells from human omental tissue. In this study, we compared the responsiveness to EGF of late passaged (senescent) human omental microvascular endothelial (HOME) cells with that of early passaged (young) HOME cells. We have employed HOME cells derived from surgically resected omental samples from 14 patients. EGF stimulated cell migration significantly more in the young cells than in the senescent cells during serial cultivation (aging) in vitro. Scatchard analysis demonstrated that the number for both high and low affinity receptors for EGF in HOME cells was decreased dramatically during serial cultivation. The expression of EGF receptor mRNA was also decreased in the senescent HOME cells. Treatment of HOME cells with EGF significantly increased cellular mRNA levels of tissue‐type plasminogen activator, and two protooncogenes, c‐fos and c‐myc, in young HOME cells, but not in senescent HOME cells. Thus HOME cells aged in vitro show a decreased responsiveness to EGF, resulting in decreased migration of human endothelial cells. The serial cultivation of human endothelial cells in vitro may downregulate EGF receptor and decrease responsiveness to exogenous EGF, a potent angiogenic factor.
Identification of estrogen receptor mRNA and the estrogen modulation of parathyroid hormone‐stimulated cyclic AMP accumulation in opossum kidney cells
Authors: Stock John L. , Coderre James A. , Burke Elizabeth M. , Danner David B. , Chipman Stewart D. , Shapiro Jay R.
Published: February 2005

The opossum kidney (OK) cell was used as a model to test the hypothesis that estrogen directly affects proximal renal tubular epithelial cells. To demonstrate the expression of estrogen receptor in OK cells, we developed an approach using reverse transcription and the polymerase chain reaction. Analysis of the DNA amplified with nested primers revealed the predicted size fragment and restriction enzyme digestion products. To demonstrate the functional effects of estrogen, OK cells at confluence were preincubated in serum‐free medium for 7–10 days with or without 17β‐estradiol. Bovine PTH(1–34) (bPTH(1–34)) then stimulated a dosedependent intracellular accumulation of cAMP that was maximal after 1 min and then gradually declined. Cyclic AMP in the medium slowly increased over 60 min. Preincubation with 17β‐estradiol did not affect cell proliferation as measured by total protein content but caused an inhibition of bPTH(1–34)‐stimulated intracellular cAMP accumulation that was maximal at 10−11M 17β‐estradiol (71 ± 3% control, p < .001). bPTH(1–34) also increased cAMP release into the medium, an effect maximal using 10−10M 17β‐estradiol (118 ± 3% control, p < .001). Preincubation with the inactive isomer 17α‐estradiol caused no changes in cAMP accumulation or release. Coincubation with the antiestrogen tamoxifen blocked the effects of 17β‐estradiol. Sodium‐dependent phosphate transport was: (1) inhibited by 2‐h incubations with 10−8 or 10−10 M bPTH(1–34) and not affected by preincubation with 17β‐estradiol, and (2) not inhibited by a 20‐min incubation with 10−8 M bPTH(1–34) unless cells were preincubated with 10−8 M 17β‐estradiol, suggesting that any possible effects of estrogen on phosphate transport are not directly mediated by changes in cAMP. These studies demonstrate the presence of estrogen receptor mRNA in OK cells as well as direct and specific effects of physiologic concentrations of estrogen on cAMP accumulation in these cells. This system may be a good model for further study of estrogen and PTH effects on the kidney.
Contrasting effects of the protein kinase C inhibitor staurosporine on the interleukin‐1 and phorbol ester activation pathways in the EL4–6.1 thymoma cell line
Authors: Dornand Jacques , Bouaboula Monsif , D'Angeac Arnaud Dupuy , Favero Jean , Shire David , Casellas Pierre
Published: February 2005

EL 4–6. 1 cells, variants of the murine EL4 thymoma cell line, can be activated by interleukin 1 (IL‐1) or phorbol 12‐myristate‐13‐acetate (PMA), or PMA + IL‐1 to secrete interleukin 2 (IL‐2) and interleukin 4 (IL‐4) and to express the IL‐2 receptor (IL‐2R). To compare the different activation pathways, we examined the effects of staurosporine (STAR) and 1‐(5‐isoquinolinylsulfonyl)‐2‐methylpiperazine (H7), two protein kinase C (PKC) inhibitors, on the induction of interleukin secretion and IL‐2R expression in these cells. We report here that nanomolar concentrations of STAR strongly potentiated (20‐ to 30‐fold) the production of IL‐2 or IL4, when EL 4–6.1 cells were induced by IL‐1 α (or IL‐1 β) alone. By contrast, at identical concentrations, STAR dose‐dependently inhibited the production of IL‐2 and IL‐4 resulting from PMA or PMA + IL‐1 cell treatment. STAR also negatively affected the expression of IL‐2R, which was dependent on PMA‐sensitive PKC with either IL‐1, PMA, or PMA + IL‐1 stimulation. The changes in interleukin production and IL‐2R expression in EL 4–6.1 activated cells were correlated with changes at the mRNA level measured by quantitative polymerase chain reaction (PCR). This finding suggests a pretranslational effect of the drug. At micromolar concentrations, H7 showed the same effects as STAR, but only increased IL‐1‐triggered interleukin secretions twofold. We observed that the action of PKC inhibitors did not result from modification of IL‐1 receptor (IL‐1R) expression in EL 4–6.1 cells. Thus, our data show that PKC inhibitors clearly distinguish between IL‐1 and PMA stimulatory pathways. In addition, they suggest that the IL‐1 stimulatory pathway involves PKC(s) [or other undefined kinase(s)] which regulate this pathway and differ from PKC(s) activated by PMA. © 1992 Wiley‐Liss, Inc.
Importance of tyrosine phosphatases in the effects of cell‐cell contact and microenvironments on EGF‐stimulated tyrosine phosphorylation
Authors: Mansbridge J. N. , Knüchel R. , Knapp A. M. , Sutherland R. M.
Published: February 2005

We have compared the EGF responses of A431 cells when grown as monolayers at a variety of cell densities of as multicellular spheroids in order to investigate the effects of cell contact and 3‐dimensional structure on signal transduction. Proliferation of the A431 squamous carcinoma cell line grown in our laboratory was unaffected by EGF when grown in monolayer culture. As 3‐dimensional, multicellular spheroids, however, growth was stimulated by EGF. The maximum volume attainable in the presence of EGF was more than 30 times that in its absence. EGF‐dependent tyrosine phosphorylation was compared under these conditions by immunohistochemistry and Western blotting. In initial experiments using published procedures, tyrosine phosphorylation was density‐dependent in monolayers and undetectable in spheroids. However, the density‐dependence was abolished by the addition of high concentrations of protein tyrosine phosphatase inhibitors (1 mM Zn++ and VO43−). The density dependence of EGF‐stimulated tyrosine phosphorylation in monolayers was, therefore, largely the result of changes in phosphatase activity rather than kinase. Using high concentrations of phosphatase inhibitors, phosphotyrosine was clearly visible by immunohistochemistry in the outermost cells of spheroids, but it was still not visible in the spheroid center. The lack of response within the spheroid was not related to the presence of EGF receptor nor diffusion of EGF. In companion experiments, we showed that staining for EGF receptor was present homogeneously throughout the spheroid and that EGF penetrated to its center under the conditions of the experiment. Thus, although an increase in tyrosine phosphatase activity was a major factor affecting tyrosine phosphorylation in the outer cells, other factors were important in the inner cells. We concluded that an increase of tyrosine phosphatase activity was the most important component of the adaptation of the EGF signal transduction system to high cell density in monolayer cultures. In spheroids, tyrosine phosphatases are also enhanced, but other factors, such as autocrine synthesis of TGF‐α and possibly the cellular distribution of EGF receptors and cell shape, play a role. © 1992 Wiley‐Liss, Inc.
Endothelium‐derived relaxing factor contributes to the regulation of endothelial permeability
Authors: Oliver Juan A.
Published: February 2005

To determine whether endothelium‐derived relaxing factor (EDRF) contributes to the regulation of endothelial permeability, the transendothelial flux of 14C‐su‐crose, a marker for the paracellular pathway across endothelial monolayers (Oliver, J. Cell. Physiol. 145:536–548, 1990), was examined in monolayers of bovine aortic endothelial cells grown on collagen‐coated filters. The permeability coefficient of 14C‐sucrose was significantly decreased by 10−3 M 8‐Bromoguanosine 3′,5′‐cyclic monophosphate or by 5 × 10−6 M glyceryl trinitrate, an activator of soluble guanylate cyclase. Depletion of L‐arginine from endothelial monolayers increased 14C‐sucrose permeability from 3.21 ± 0.59 to 3.88 ± 0.50 × 10−5 cm · sec−1 (mean ± SEM; n = 6; P < 0.05). The acute administration of 5 × 10−4 M L‐arginine to monolayers depleted of this amino acid decreased 14C‐sucrose permeability from 2.91 ± 0.27 to 2.52 ± 0.26 × 10−5 cm · sec−1 (n = 11; P < 0.05). 14C‐sucrose permeability was increased by 10−7 M bradykinin and this effect was enhanced by the presence of each one of the following compounds: 10−5 M methylene blue, 4 × 10−6 M oxyhemoglobin, 5 × 10−4 M NG‐methyl‐L‐arginine or 5 × 10−4 M Nω‐nitro‐L‐arginine. These results suggest that EDRF contributes to the sealing of the endothelial monolayer and that EDRF released by bradykinin acts as a feedback inhibitor attenuating the increase in endothelial permeability induced by this peptide. Because endothelial cells have the ability to contract and relax and possess guanylate cyclase responsive to nitric oxide, our results suggest that EDRF decreases 14C‐sucrose permeability by relaxing endothelial cells, thereby narrowing the width of endothelial junctions. © 1992 Wiley‐Liss, Inc.
Inhibition by 1,25 dihydroxyvitamin D3 of c‐myc down‐regulation and DNA fragmentation in cytosine arabinoside–induced erythroid differentiation of K562 cells
Authors: Moore Dorothy C. , Carter Darryl L. , Studzinski George P.
Published: February 2005

The effect of 1,25‐dihydroxyvitamin D3 (1,25(OH)2D3) on DNA fragmentation, altered expression of the heat shock protein (hsp) 70 gene, and protooncogenes c‐myc and c‐myb was studied during chemical induction of erythroid differentiation in K562 cells. Preincubation of K562 cells with 1,25(OH)2D3 did not alter the concentration of hemoglobin in cells which did differentiate, but led to a reduction in the accumulation of low molecular weight DNA generated by Ara‐C administration. The extent of this reduction was similar to the degree of inhibition of hemoglobin formation in the culture as the whole. Preincubation with 1,25(OH)2D3 had no effect on the increase of hsp 70 gene expression induced by a 48‐hr treatment with Ara‐C, but prevented the Ara‐C‐induced down‐regulation of the protooncogene c‐myc. The protooncogene c‐myb was down‐regulated after 15 min of treatment with Ara‐C, and exposure to 1,25(OH)2D3 prior to Ara‐C caused a further down‐regulacion of its expression. The data suggest that the events associated with erythroid differentiation may be separable into at least two groups; one of these may have an influence on the kinetics of the cell cycle traverse, and the other may be related to the expression of the erythroid phenotype. © 1992 Wiley‐Liss, Inc.
Induced thermotolerance to apoptosis in a human T lymphocyte cell line
Authors: Mosser Dick D. , Martin Luis H.
Published: February 2005

A brief exposure to elevated temperatures elicits, in all organisms, a transient state of increased heat resistance known as thermotolerance. The mechanism for this thermotolerant state is unknown primarily because it is not clear how mild hyperthermia leads to cell death. The realization that cell death can occur through an active process of self destruction, known as apoptosis, led us to consider whether thermotolerance provides protection against this mode of cell death. Apoptosis is a common and essential form of cell death that occurs under both physiological and pathological conditions. This mode of cell death requires the active participation of the dying cell and in this way differs mechanistically from the alternative mode of cell death, necrosis. Here we show that mild hyperthermia induces apoptosis in a human leukemic T cell line. This is evidenced by chromatin condensation, nuclear fragmentation and the cleavage of DNA into oligonucleosome size units. DNA fragmentation is a biochemical hallmark of apoptosis and requires the activation of an endogenous endonuclease. The extent of DNA fragmentation was proportional to the severity of heat stress for cells heated at 43°C from 30 to 90 minutes. A brief conditioning heat treatment induced a resistance to apoptosis. This was evident as a resistance to DNA fragmentation and a reduction in the number of apoptotic cells after a heat challenge. Resistance to DNA fragmentation developed during a recovery period at 37°C and was correlated with enhanced heat shock protein (hsp) synthesis. This heat‐induced resistance to apoptosis suggests that thermotolerant cells have gained the capacity to prevent the onset of this pathway of self‐destruction. An examination of this process in heated cells should provide new insights into the molecular basis of cellular thermotolerance. © 1992 Wiley‐Liss, Inc.
Regulation of vascular smooth muscle cell integrin expression by transforming growth factor β1 and by platelet‐derived growth factor‐BB
Authors: Janat M. Fouad , Argraves W. Scott , Liau Gene
Published: February 2005

We have examined the ability of transforming growth factor‐β1 (TGF‐β1) and platelet‐derived growth factor‐BB (PDGF‐BB) to regulate the expression of various integrins in cultured rabbit vascular smooth muscle cells (SMC). We found that expression of the αvβ3 integrin complex was induced by both growth factors, although TGF‐β1 appeared to be the more potent inducer. mRNA level of the β3 integrin subunit was undetectable in quiescent cells and enhanced by both growth factors, while the αv integrin subunit mRNA level did not change with growth factor addition. Therefore, appearance of the αvβ3 integrin protein complex after growth factor stimulation was due to increased expression of the β3 integrin subunit mRNA. The TGF‐β1 induced increase in β3 integrin mRNA was delayed, but did not require prior protein synthesis, since cycloheximide was unable to block the increase in β3 mRNA level. By contrast, PDGF‐BB induced a more rapid increase in β3 integrin mRNA level that peaked by 6 h after growth factor addition and no detectable β3 integrin mRNA remained after 24 h. Interestingly, the PDGF‐BB induced elevation of β3 integrin, although more rapid, was completely inhibited by cycloheximide. Expression of the α5 integrin subunit in response to growth factors was very similar to β3. However, in contrast to β3 and α5, neither TGF‐β1 nor PDGF‐BB were able to alter the expression of the β1 integrin subunit in vascular SMC. However, in TGF‐β1 treated cells, there was a large increase in expression of a 190 kDa polypeptide that was associated with the β1 integrin subunit. This 190 kDa polypeptide was not detected in PDGF treated SMC or in TGF‐β1 treated fibroblasts. The α1 integrin subunit has a MW of approximately 190 kDa and is capable of complexing with β1. Analysis of the α1 integrin subunit mRNA level indicated that it was indeed induced by TGF‐β1, but not by PDGF‐BB, suggesting that the 190 kDa polypeptide may be the α1 integrin subunit. These results indicate that TGF‐β1 and PDGF‐BB are potent but distinct activators of integrin expression in vascular SMC. © 1992 Wiley‐Liss, Inc.
Ca2+ accumulation and loss by aberrant endocytic vesicles in sickle erythrocytes
Authors: Williamson Patrick , Puchulu Estela , Penniston John T. , Westerman Maxwell P. , Schlegel Robert A.
Published: February 2005

Sickle cells contain internal vesicles which accumulate Ca2+. As shown here, the membrane enclosing the vesicles contains the plasma membrane Ca2+‐ATPase, or Ca2+ pump, as judged by staining with an antibody directed against the protein. Moreover, the number of cells containing such vesicles increases upon deoxygenation. These findings argue strongly that the vesicles arise by endocytosis from the plasma membrane, and explain how they accumulate Ca2+. When sickle cells are depleted of ATP Ca2+ is lost from the vesicles, as judged by the disappearance of staining with the Ca2+/membrane probe chlortetracycline (CTC), without a corresponding loss of antibody staining. This loss of Ca2+ can be inhibited by nitrendipine, a Ca2+ channel blocker. These results suggest that the vesicle membrane allows outward passage of Ca2+ by a nitrendipine‐sensitive pathway, which can be overcome by the inward‐directed activity of the Ca2+ pump of the vesicle membrane. If so, the Ca2+ which vesicles contain is in dynamic equilibrium with the cytoplasm of the sickle erythrocyte. © 1992 Wiley‐Liss, Inc.
Effects of acute vs. chronic phorbol ester exposure on transepithelial permeability and epithelial morphology
Authors: Mullin James M. , Snock Kathleen V. , Shurina Robert D. , Noe Jasna , George Kenneth , Misner Lisa , Imaizumi Shotaro , O'Brien Thomas G.
Published: February 2005

In previous experiments we have shown that acute (30 minutes) exposure to phorbol esters or other protein kinase C activators causes increased transepithelial permeability, specifically by the increased paracellular permeability through tight junctions. However, the role of protein kinase C activators in carcinogenesis is predicted upon a chronic exposure of an effective dose at frequent intervals for a prolonged period of time. We therefore sought to determine the effect of chronic phorbol ester exposure on transepithelial permeability by exposing cells of the polar renal epithelial cell line, LLC‐PK1, to phorbol esters for time periods as long as 16 weeks. The following changes ensued: (1) after the initial drop in transepithelial resistance due to phorbol ester exposure, i.e., an increase in transepithelial permeability (in the acute phase of exposure), an adaptive response occurs as transepithelial resistances in chronically exposed cultures recover to approximately 50% of control values, (2) the cell sheets in chronically exposed cultures lose their acute responsiveness of transepithelial permeability to phorbol ester exposure, (3) cell sheet architecture changes as cells occasionally multilayer and actual polyp‐like cell masses appear at high frequency, and (4) cytosolic protein kinase C activity decreases to 50% of control level with acute exposure and then is further decreased to less than 1% of control level in chronically treated cells; membrane‐associated PKC activity is not as sharply decreased. The possible role of transepithelial permeability in carcinogenesis and the value of chronically treated epithelial cell cultures as a model for two‐stage carcinogenesis are discussed. © 1992 Wiley‐Liss, Inc.
Thrombin and histamine rapidly stimulate the phosphorylation of the myristoylated alanine‐rich C‐kinase substrate in human umbilical vein endothelial cells: Evidence for distinct patterns of protein kinase activation
Authors: Jacobson Brian C. , Pober Jordan S. , Fenton John W. , Ewenstein Bruce M.
Published: February 2005

Human α‐thrombin and histamine each stimulates protein phosphorylation in human umbilical vein endothelial cells (HUVEC). We have identified the most prominent of these phosphoproteins by immunoprecipitation as the human homolog of the widely distributed myristoylated alanine‐rich C‐kinase substrate (MARCKS). Stimulation by 0.1–10 U/ml of α‐thrombin produces a time‐dependent, sustained (plateau 3–5 min) level of MARCKS phosphorylation. MARCKS phosphorylation requires thrombin catalytic activity but not receptor binding and is also seen in response to stimulation by a peptide, TR (42–55), that duplicates a portion of the thrombin receptor tethered ligand created by thrombin proteolytic activity. One micromolar histamine, like α‐thrombin, produces sustained phosphorylation of MARCKS (plateau 3–5 min). In contrast, 100 μM histamine results in rapid but transient MARCKS phosphorylation (peak 1–3 min). HUVEC treated with 100 μM histamine for 5 min can be restimulated by α‐thrombin but not fresh histamine, suggesting that the histamine receptor was desensitized. MARCKS phosphorylation can also be induced by several exogenous protein kinase C (PKC) activators and both α‐thrombin‐ and histamine‐induced MARCKS phosphorylation are inhibited by the PKC antagonist staurosporine. However, while prolonged PMA pretreatment ablates histamine‐induced MARCKS phosphorylation, the ability of thrombin to induce MARCKS phosphorylation is retained. These findings provide evidence for agonist‐specific pathways of protein kinase activation in response to thrombin and histamine in HUVEC. © 1992 Wiley‐Liss, Inc.
Calcium alginate beads as a slow‐release system for delivering angiogenic molecules In Vivo and In Vitro
Authors: Downs Elizabeth C. , Robertson Nancy E. , Riss Terry L. , Plunkett Marian L.
Published: February 2005

A method previously used in this laboratory for entrapment of tumor cells in alginate beads has been extended to provide a slow release delivery system for growth factors with known in vivo angiogenic activity. Protein growth factors were entrapped in alginate beads in amounts sufficient to cause incorporation of 3H‐thymidine by COMMA‐D cells in vitro, and in vivo neovascularization when injected subcutaneously into Balb/c mice. Entrapment of 125I‐labelled growth factors showed that the amount of molecule entrapped in alginate beads may vary with the charge of the molecule. In vitro cell proliferation studies showed that entrapment in alginate beads may provide a slow‐release system or a stabilizing environment for the protein. In some cases biological activity of the growth factor in solution was increased by the presence of control alginate beads. When alginate‐entrapped growth factors were injected into Balb/c mice, induction of new blood vessels could be monitored qualitatively by macroscopic photography and assessed quantitatively by measuring the pooling of radiolabelled red blood cells at the experimental site. Subcutaneous injection of purified angiogenic factors not entrapped in alginate beads did not cause neovascularization. Diffusion of 125I‐labelled growth factors from alginate beads in the animal showed that release in vivo may depend on the charge of the protein molecule. These results indicate that injection of purified molecules entrapped in alginate beads provides an effective localized and slow‐release delivery of biologically active molecules. This delivery system may extend the time of effectiveness of biologically active molecules in vivo compared to direct injection without alginate entrapment. The method of entrapment and injection has potential for identifying active factors in tumor‐induced angiogenesis and testing new compounds as modulators of neovascularization. © 1992 Wiley‐Liss, Inc.
Ethanol inhibits ligand‐activated Ca2+ channels in human B lymphocytes
Authors: Brodie Chaya , Domenico Joanne , Mazer Bruce D. , Gelfand Erwin W.
Published: February 2005

Ethanol reportedly is immunosuppressive, interfering with lymphocyte proliferation. To investigate the basis for this immunosuppression, the effects of acute treatment with ethanol were studied on Ca2+ mobilization in tonsillar B lymphocytes and the human lymphoblastoid B‐cell line, Ramos. The level of intracellular Ca2+ was monitored in cells loaded with the fluorescent dye indo‐1 following stimulation with either anti‐lgM antibody or platelet activating factor. The effect of ethanol was also examined on the induction of the early proto‐oncogene c‐fos in these cells. Ethanol inhibited ligand‐activated Ca2+ mobilization due to transmembrane influx but not intracellular store release, in a dose‐and time‐dependent manner. This inhibition was not due to the inability of anti‐lgM to bind to its surface receptor nor to membrane depolarization induced by ethanol. Ethanol also inhibited the Ca2+‐dependent induction by anti‐lgM of c‐fos in these cells. The inhibitory effects of ethanol on ligand‐activated Ca2+ channels and subsequent induction of c‐fos may provide the basis for its immunosuppressive action. © 1992 Wiley‐Liss, Inc.
EGF receptor regulation in normal mouse mammary gland
Authors: Haslam Sandra Z. , Counterman Laura J. , Nummy Katherine A.
Published: February 2005

Estrogen (E), progesterone (P), and epidermal growth factor (EGF) are known to regulate growth and development of the normal mammary gland, and it is possible that EGF may interact with E and/or P. Estrogen (ER), progesterone (PR), and EGF receptors (EGF‐R) have been detected in both mammary epithelial and stromal cells, and the relative roles of the various cells types in hormone‐dependent growth regulation are not known. The present studies were undertaken to determine if E and/or P influence EGF action by exerting a regulatory effect on EGF‐R levels and which cell types are affected. The comparative effects of ovariectomy and hormone treatments on EGF‐R levels were examined in immature, pubertal 5‐week‐old and sexually mature 10‐week‐old female mice. EGF‐R were characterized as a single class of high affinity sites and EGF‐R concentration was 2‐fold higher in glands of 5‐week‐old mice. Ovariectomy had no significant effect on EGF‐R concentration in either age group, and treatment with E and/or P had no effect on EGF‐R levels in either epithelial or stromal cells in 5‐week‐old mice. In contrast, E+P treatment caused a 2‐fold increase in receptor concentration in 10‐week‐old mice in the mammary epithelium. Thus it appears that the developmental state of the gland may determine the nature and extent of the interaction of EGF, E, and P. © 1992 Wiley‐Liss, Inc.
Amnion cell biosynthesis of interleukin‐8: Regulation by inflammatory cytokines
Authors: Trautman Michael S. , Dudley Donald J. , Edwin Samuel S. , Collmer Debbie , Mitchell Murray D.
Published: February 2005

The cellular constituents of the placenta are important participants in the recruitment and trafficking of inflammatory cells within the placenta. In infectioninduced labor, gestational tissues synthesize and release a variety of inflammatory cytokines whose effects include increased prostaglandin biosynthesis and the initiation of uterine contractions. Interleukin‐8 (IL‐8), a potent neutrophil chemoattractant, has been recently described as being elevated in the amniotic fluid of mothers with chorioamnionitis. We investigated the biosynthesis of IL‐8 by human amnion cells and its regulation by other inflammatory cytokines. Cultured amnion cells obtained from normal term placentae were found to produce IL‐8 in response to pathophysiologic concentrations of interleukin 1β (IL‐1β) and tumor necrosis factor alpha (TNF‐α). Treatment of amnion cells stimulated by IL‐1β with cycloheximide resulted in increased IL‐8 production, while incubation of IL‐1β treated amnion cells with actinomycin D resulted in a concentration‐dependent decrease in detectable amounts of IL‐8. Northern blot analysis of cultured amnion cells stimulated with IL‐1β demonstrated a rapid increase in IL‐8 mRNA which peaked at 2–4 hr. These in vitro result suggest inflammation of gestational tissues in vivo may result in locally produced IL‐8 and, in association with other inflammatory mediators, may be important in the pathophysiology of infection‐induced labor. © 1992 Wiley‐Liss, Inc.
Neutrophil adhesion to TNFα‐activated endothelial cells potentiates leukotrine B4production
Authors: Ishii Yoshiki , Lo Siu K. , Malik Asrar B.
Published: February 2005

Since adhesion of neutrophils (PMN) to endothelial cells may influence PMN activation responses, we examined whether adhesion of PMN to TNFα‐activated human umbilical vein endothelial cells (HUVEC) stimulates leukotriene B4(LTB4) production. Endothelial adhesivity towards PMN increased after HUVEC pretreatment with TNFαfor 4 h. LTB4production increased markedly in response to stimulation with arachidonic acid (20 μM) when PMN were added to the hyperadhesive HUVEC. In contrast, stimulation of PMN in suspension did not potentiate LTB4production. LTB4production persisted when PMN were applied to TNFα‐pretreated HUVEC fixed with 1% paraformaldehyde excluding the possibility that metabolic activity of endothelium participates in this response. PMN adhesion to plastic and gelatin also enhanced LTB4indicating that adhesion was critical event in inducing LTB4production. We used monoclonal antibodies (mAb) to adhesion molecules on endothelial cells (i.e., endothelial leukocyte adhesion molecule‐1 (ELAM‐1) and intercellular adhesion molecule‐1 (ICAM‐1) or on PMN (CD18) to assess the role of PMN adhesion to the activated endothelium on LTB4potentiation. Both anti‐ELAM‐1 mAb and anti‐ICAM‐1 mAb inhibitied PMN adhesion (by 55 and 41% respectively) as well as LTB4production (by 65 and 50% respectively). Anti‐CD18 mAB also reduced the adhesion (65%) and the LTB4production (66%). Furthermore, combination of anti‐ELAM‐1 mAb (H18/7) and anti‐ICAM‐1 mAb (RR1/1) or of anti‐ELAM‐1 mAb (H18/7) and anti‐CD 18 mAb (IB4) had an additive effect in inhibiting both PMN adhesion as well as LTB4production. PMN adherence to immobolized recombinant soluble rELAM‐1 or rlCAM‐1 also increased LTB4production, which was prevented with relevant mAbs. However, neither rELAM‐1 nor rlCAM‐1 stimulated LTB4production of PMN in suspension. We conclude that PMN adhesion to TNFα‐stimualted endothelial cells enhances LTB4production by PMN, a response activated by binding of PMN to expressed endothelial cell surface adhesion molecules. © 1992 Wiley‐Liss, Inc.
Tributyltin is a potent inducer of the heat shock response in human diploid fibroblasts
Authors: Zhang Hua , Liu Alice Y.‐C.
Published: February 2005

Submicromolar concentrations of tributyltin (TBT), a commercially used organotin compound, were found to induce the expression of several stress proteins, most notably HSP89 and HSP70, in IMR‐90 human diploid fibroblats in a time‐ and dose‐dependent manner. This induction can be demonstrated by quantitation of 1) synthesis of the heat shock proteins (HSPs), 2) relative abundance of mRNA of hsp70, and 3) transient expression of a human hsp70 promoter driven reporter gene. TBT also increased the abundance of mRNA of heme oxygenase, whereas heat shock was without effect. Analysis of protein binding to a consensus heat shock element (HSE)by electrophoretic mobility shift assay suggests that the induction of the heat shock response by TBT was attributable to activation of the heat shock transcription factor (HSTF). © 1992 Wiley‐Liss, Inc.
Inhibitory effects of a bacteria‐derived sulfated polysaccharide against basic fibroblast growth factor‐induced endothelial cell growth and chemotaxis
Authors: Nakayama Yutaka , Iwahana Michio , Sakamoto Noritsugu , Tanaka Noriko G. , Osada Yasuaki
Published: February 2005

The effects of sulfated polysaccharides on the growth and chemotaxis of endothelial cells promoted by basic fibroblast growth factor (bFGF), a heparin‐binding growth factor, and epidermal growth factor (EGF), a non‐heparin‐growth factor, were examined. The binding abilities of these two growth factors to D‐gluco‐galactan sulfate (DS‐4152) were the same as to heparin. DS‐4152 inhibited the growth and chemotaxis of the cells stimulated by bFGF, and prevented the binding of bFGF to the cells at both its low and high affinity binding sites: the former and the latter are heparin‐like molecules and receptor proteins for bFGF, respectively. However, DS‐4152 affected neither the binding of EGF to endothelial cells nor the proliferation and chemotaxis of the cells stimulated by the factor. Heparin also inhibited the binding of bFGF to low affinity binding sites to the same degree as DS‐4152, but had little effect on the binding of bFGF to high affinity sites and no effects on bFGF‐induced endothelial cell growth. Chondroitin sulfate A prevented neither the binding of bFGF to both sites of the cells nor bFGF‐induced cell proliferation. We thus concluded that the inhibitory effects of DS‐4152 against the growth and chemotaxis of endothelial cells induced by bFGF might be due to the prevention of bFGF binding to its receptor proteins resulting from the binding of DS‐4152 to bFGF. © 1993 Wiley‐Liss, Inc.
Differential antimitogenic effectiveness of atrial natriuretic peptides in primary versus subcultured rat aortic smooth muscle cells: Relationship to expression of ANF‐C receptors
Authors: Cahill Paul A. , Hassid Aviv
Published: February 2005

Previous studies have shown that atrial natriuretic peptides inhibit mitogenesis in subcultured aortic smooth muscle cells by a mechanism that appears to be mediated via the C‐type or “clearance” receptor. In the current study, we have compared the antimitogenic effect of these peptides in serum‐stimulated primary aortic smooth muscle cell cultures and in subcultured cells. A series of atrial peptides, including rANF99–126, rANF103–126, and rANF103–125, were only poorly antimitogenic in serum‐stimulated primary cultures, whereas des[Cys105, Cys121] rANF104–126 which binds selectively to the ANF‐C receptors had no antimitogenic activity. In contrast, in subcultured cells (between subcultures 3 and 25), rANF99–126, rANF103–126, rANF103–126, Cys116rANF102–116, and des[Cys105, Cys121]rANF104–126 inhibited serum‐induced [3H]thymidine incorporation (IC50 in the range of 10–50 nM), with maximal inhibition of 40–70%. The lack of antimitogenic activity in primary cultures did not appear to be related to the lack of cGMP elevation elicited by atrial peptides or to an inherent insensitivity to the action of antimitogens, because primary cultures were responsive to the cGMP‐elevating effect of atrial peptides and the cells were more rather than less sensitive to the antimitogenic effect of the nitric‐oxide‐vasodilator, SNAP, as compared to subcultured cells. Analysis of the affinity and binding capacity of freshly isolated aortic membranes, and primary or secondary cultures for [125I]rANF99–126, revealed that the number of ANF receptors increased by tenfold, following subculture. Moreover, subcultured cells contained receptors with increased binding affinity for peptide analogues selective for the ANF‐C‐type type receptor. Covalent cross‐linking studies with (125I)rANF99–126 confirmed that membranes prepared from fresh aortae predominantly expressed the ANF‐A/guanylate cyclase receptor, whereas in subcultured cells the predominantly cross‐linked protein was the ANF‐C‐type receptor, with receptors in primary cultures occupying an intermediate position. These results suggest that the binding and antimitogenic activity of atrial peptides in aortic smooth muscle cells depends on the phenotypic state of these cells. Moreover, the increased antimitogenic potency of atrial peptides in secondary cultures may reflect increased expression of the ANF‐C‐type receptors. © 1993 Wiley‐Liss, Inc.
Contrasting effect of transforming growth factor type beta 1 (TGF‐β1) on proliferation and interleukin‐2 receptor expression in activated and rapidly cycling immature (CD3−CD4−CD8−) thymocytes
Authors: d'Angeac Arnaud Dupuy , Rème Thierry , Monier Serge , Gao Qinglin , Duperray Christophe , Jullien Pierre , Dornand Jacques
Published: February 2005

Transforming growth factor β (TGF‐β) is a cytokine with immunoregulatory properties that acts negatively on T lymphocyte proliferation. However, with the EL 4–6.1 variant of the murine thymoma EL 4 activated with phorbol ester and/or interleukin‐1 (IL‐1), we recently found that it up‐regulates interleukin‐2‐receptor (IL‐2R) expression. Since EL 4–6.1 cells share phenotypic and functional characteristics with the immature thymic subset lacking CD4 and CD8 accessory molecules (DN), we investigated the effect of TGF‐β1 on the IL‐2R 55kD α chain expression and proliferation of activated DN cells and especially in DN cells that do not express CD3. We observed that TGF‐β1 was able to increase both the percentage of CD3−DN cells expressing IL‐2Rα chains and the expression of IL‐2Rα chain in these cells. This stimulatory effect of TGF‐β1 was distal from early transduction events. In addition, TGF‐β1 was found to modulate CD3−DN cell proliferation. During differentiation in the thymus, CD3−DN cells transiently express the IL‐2Rα chain of the IL‐2R and these IL‐2R+ CD3−DN cells are preprogrammed to down‐regulate the IL‐2Rα chain and up‐regulate the CD4 and CD8 accessory molecule. We thus also tested the effect of TGF‐β1 on IL‐2Rα chain expression in these in vitro differentiating CD3−DN cells. We found that TGF‐β1 neither significantly affected IL‐2R expression nor changed CD4 or CD8 expression. Hence, in CD3−DN cells, the effect of TGF‐β1 on IL‐2R expression seems to be restricted to proliferating cells. © 1993 Wiley‐Liss, Inc.
Role of receptor complexes in resistance or sensitivity to growth inhibition by TGFβ in intestinal epithelial cell clones
Authors: Mulder Kathleen M. , Segarini Patricia R. , Morris Sheila L. , Ziman Jill M. , Choi Ho Gene
Published: February 2005

Untransformed rat intestinal epithelial cells (IEC‐18) were chemically mutagenized, selected in the presence of TGFβ1, and cloned by limiting dilution. Two clones (4–5, 4–6) were resistant to growth inhibition by both TGFβ1 and TGFβ2. Another clone (4–1) was more sensitive to both TGFβ isoforms (relative to parental IEC‐18 cells). IC50 values for TGFβ1 and 2 in the 4–1 cells were at least 1/9 those of the parental cells; growth rates were reduced by 49% for TGFβ1 and by 26% for TGFβ2 in this clone. This increased sensitivity to TGFβ was explained by the 5‐ to 10‐fold increase, relative to parental cells, in binding of TGFβ1 and TGFβ2 to both the type I and II receptors. In contrast, the resistance to growth inhibition by TGFβ in the 4–5 and 4–6 cells could not be explained by a decrease in either TGFβ binding affinities or in total number of receptors expressed, by the presence of serum binding components, or by occupation of receptor binding sites with autocrine TGF‐β1. However, in comparison to TGFβ‐sensitive cells (IEC‐18, 4–1), the resistant cells displayed a higher ratio of type II relative to type I receptor binding by TGF‐β1. Thus, a critical ratio of binding to receptor subtypes correlated with growth inhibition by TGF‐β1. Resistance to TGF‐β2 in the same clones did not appear to be receptor related. Thus different mechanisms for resistance to TGF‐β1 and TGF‐β2 were observed within a given clone. © 1993 Wiley‐Liss, Inc.
19F NMR studies of changes in membrane potential and intracellular volume during dexamethasone‐induced apoptosis in human leukemic cell lines
Authors: Adebodun Foluso , Post Jan F. M.
Published: February 2005

The induction of apoptosis in leukemic cells by dexamethasone is well known, but the mechanism of this type of cell death and of dexamethasone resistance by some variants is still poorly understood. Apoptotic cell death is preceded by many changes in cellular properties, such as glucose metabolism, cell size, cell density, and others. In this study, 19F‐NMR has been used to characterize changes in cell membrane potential and intracellular accessible volume during dexamethasone induced apoptosis. One dex‐sensitive (CEM‐C7) and three dex‐resistant variants (CEM‐C1, CEM‐ICR27, and CEM‐4R4) were examined. We have observed separate intracellular and extracellular resonances for trifluoroacetate and trifluoroacetamide added to suspended leukemic cells. From the equilibrium distribution of these fluoro‐compounds between intra and extracellular spaces, the changes in membrane potential and intracellular accessible volume were calculated. The membrane potential for CEM‐C7 cells was found to significantly decrease in the presence of dexamethasone (9‐mV decrease within 18 h of dexamethasone treatment), while that of CEM‐ICR27 was found in some samples to increase on dexamethasone incubation. The membrane potential for CEM‐C1 decreased slightly, while that of CEM‐4R4 was not appreciably affected by dexamethasone. The reduction of membrane potential seems to be an early step in the mechanism of dexamethasone induced apoptosis. Although the intracellular volume varied with cell type and dexamethasone incubation (for CEM‐C7), the fractional intracellular volume (α = Vin/Vcell was found to be the same (0.82 ± 0.06) for all the cell lines in the presence and absence of dexamethasone. © 1993 Wiley‐Liss, Inc.
Macrophage supernatants have both stimulatory and suppressive effects on mesangial cell proliferation
Authors: Mattana Joseph , Singhal Pravin C.
Published: February 2005

Macrophages may modulate mesangial expansion following renal injury via secretory products. We undertook the present study to determine the effects of macrophages supernatants on mesangial cell proliferation. Macrophage supernatants collected in serum‐free media after 24 hours caused significantly enhanced mesangial cell proliferation in long‐term culture at concentration up to 50% but caused suppression at higher concentration (control, 122,000 ± 14,000 cells/well: 50% supernatant, 188,000 ± 15,100 cells/well, p < 0.02 compared to control, n = 4; 80% supernatant, 52,000 ± 3,500 cells/well, P < 0.01 compared to control, n = 4). In short‐term culture [3H] thymidine incorporation, a measure of DNA synthesis, was significantly enhanced compared to control at supernatant concentrations up to 30% (30% supernatant, 4,120 ± 310 cpm/well; control, 3,210 ± 97 cpm/well, P < 0.5, n = 4), but uptake was reduced at high concentration (80% supernatant, 2,900 ± 74 cpm/well; control, 3,210 ± 97 cpm/well, P < 0.05, n= 4) When macrophages supernatants were collected after 48 hours incubation and incubated with mesangial cells, mesangial cell thymidine uptake was significantly suppressed compared to control (48‐hours supernatant, 4,060 ± 260 cpm/well; control, 5,890 ± 270 cpm/well, P < 0.01, n = 4) and comapared to 24‐hour supernatants, which enhanced uptake (24‐hour supernatant, 8,080 ± 340 cpm/well; control, 5,890 ± 270 cpm/well, P < 0.01, n =4). Our results suggest that macrophages supernatants can directly enhance mesangial cell proliferation in vitro in both short‐term and long‐term culture, though this effect is lost at high concentrations of supernatant. These data lend support to the potential role of the macrophage in mediating mesangial expansion following renal injury. © 1993 Wiley‐Liss, Inc.