bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparative study of the tyrosine phosphorylation of proteins in Swiss 3T3 fibroblasts stimulated by a variety of mitogenic agents
Authors: Pasquale Elena B. , Maher Pamela A. , Singer S. J.
Published: February 2005

We have carried out a comparative study of the protein tyrosine phosphorylation induced by a wide range of mitogenic stimuli on a single cell type, Swiss 3T3 mouse fibroblasts. For this purpose we have used high‐affinity antibodies directed to phosphotyrosine residues on proteins (Wang: Mol. Cell. Biol. 5:3640–3643, 1985) in immunoblotting and immunofluorescence microscopy experiments. Immunoblotting experiments showed that all of the mitogens tested, including epidermal growth factor, platelet‐derived growth factor, basic fibroblast growth factor, insulin, fetal calf serum, trypsin, and 12‐O‐tetradecanoylphorbol‐13‐acetate, increased the phosphorylation on tyrosine of a number of proteins. Most of the increase in tyrosine phosphorylation induced by each factor involved a small set of proteins with apparent molecular weights (Mr) above 50,000. Following stimulation with epidermal growth factor, platelet‐derived growth factor, and basic fibroblast growth factor, increased phosphotyrosine modification of proteins with molecular weights corresponding to those of the respective receptors was observed. A protein band of apparent Mr 160,000 contained substantially increased levels of phosphotyrosine following insulin treatment, but tyrosine phosphorylation of the insulin receptor was apparently below the level of detectability. The phosphotyrosine content of proteins with apparent Mr of 220,000, 120,000, and 70,000 was increased by all the agents tested. Phosphorylation on tyrosine of most of the proteins increased within a few minutes of the mitogenic stimulation, reached a peak, and returned more slowly to basal levels. Immunofluorescence labeling with the antibodies specific for phosphotyrosine showed a substantial increase in the amount of phosphotyrosine containing proteins only in the presence of platelet‐derived growth factor and fetal calf serum. This finding suggests that most of the proteins phosphorylated on tyrosine in Swiss 3T3 fibroblasts are not concentrated in specific subcellular structures, but rather are diffusely distributed throughout the cell and are therefore not detectable by immunofluorescence microscopy.
Enhanced constitutive expression of the 27‐kDa heat shock proteins in heat‐resistant variants from chinese hamster cells
Authors: Chrétien Pierre , Landry Jacques
Published: February 2005

Four heat‐resistant variants were isolated after treatment of Chinese hamster lung cells with the mutagen ethyl methane sulfonate, followed by a single‐step selection procedure consisting in a severe hyperthermic treatment of 4 h at 44°C. The isolated clones had a stable resistant phenotype for at least 150 generations during which they showed a 5,000‐fold increased survival to a 4‐h treatment at 44°C when compared to wild‐type cells. Comparative two‐dimensional electrophoretic analyses of proteins revealed that, like induced thermotolerant wild‐type cells (i.e., cells induced to a transient physiological state of thermotolerance by a sublethal heat conditioning treatment administered 18 h before), the heat‐resistant variants had, at normal temperature, an increased content of a heat‐shock protein with Mr of 27,000 (HSP27). In three of the four heat‐resistant variants, the increased content of HSP27 was correlated with a two‐fold increase in the constitutive level of the mRNA encoding HSP27. Chinese hamster HSP27 is composed of three species that differ in their relative isoelectric point, among which the two most acidic forms are phosphoproteins. In both the heat‐resistant variant and wild‐type cells, heat shock induces a rapid enhancement of the phosphorylation of HSP27: maximal phosphorylation occurs within 10 min upon changing the incubation temperature from 35° to 44°C. A concomitant shift in silver‐staining intensity is rapidly detectable between the three isoforms, which seems to indicate that the two phosphorylated species represent post‐translational modifications of the more basic species. It is concluded that most likely the enhanced expression of HSP27 is linked to the resistant phenotype of the variants. The study provides supporting evidence that both the content and phosphorylation status of HSP27 are determining factors in the ability of cells to survive hyperthermic treatments.
Bombesin enhancement of cAMP accumulation in swiss 3T3 cells: Evidence of a dual mechanism of action
Authors: Millar Jonathan B. A. , Rozengurt Enrique
Published: February 2005

Addition of bombesin in the presence of either forskolin or cholera toxin caused a marked (4–6 fold) enhancement of cAMP accumulation in Swiss 3T3 cells. This effect was time and concentration dependent, induced by various bombesin‐like peptides and blocked by a bombesin antagonist. Enhancement of cAMP accumulation by bombesin was diminished by chronic pretreatment with phorbol dibutyrate implicating the involvement of protein kinase C in the activation. Pretreatment with pertussis toxin, which uncouples protein kinase C activation from cAMP accumulation (Proc. Natl. Acad. Sci. U.S.A., 84:2282, 1987) also inhibited bombesin enhancement of cAMP. Bombesin was also shown to release E type prostaglandins into the medium, an effect which was abolished by the cyclooxgenase inhibitor indomethacin. Low concentrations (100 nM) of indomethacin partially inhibited the accumulation of cAMP by bombesin in the presence of forskolin indicating that the release of E type prostaglandins into the medium is also involved in the accumulation of cAMP by bombesin. The additive nature of PBt2‐mediated down‐regulation and treatment with indomethacin suggests that activation of protein kinase C and the release of E type prostaglandins provide two distinct pathways involved in the enhancement of cAMP accumulation by bombesin. Finally, bombesin in the presence of forskolin stimulated the phosphorylation of the intermediate filament component vimentin, identified in the accompanying paper as a substrate for a cAMP dependent protein kinase in intact Swiss 3T3 cells.
Regulatory volume decrease in alveolar macrophages: Cation loss is not correlated with changes in membrane recycling
Authors: Novak Jeanne M. , Cala Peter M. , Ward Diane McVey , Buys Saundra S. , Kaplan Jerry
Published: February 2005

Alveolar macrophages regain their normal volume after swelling in hypo‐osmotic solutions. This process, termed regulatory volume decrease (RVD), is initiated 3–5 minutes after exposure of cells to hypo‐osmotic solutions, and by 30 min, near‐normal volumes are attained. Volume decrease does not occur at 0°C or in solutions in which Na+ has been replaced by K+, or Cl− by the impermeant anion gluconate. These results, as well as direct measurement of intracellular cations, indicate that decrease in cell volume result primarily from the loss of K+ and Cl− and are similar to RVD in lymphocytes. Kinetic analysis of cation loss, both by directly measuring changes in intracellular cation content and by assaying rubidium efflux, showed that cation loss occurred immediately upon media dilution. The rate of cation loss fit first‐order kinetics and preceded both the initiation of volume decrease and the maximum increase in surface receptor number. These results suggest that the cation transporters responsible for RVD are located at the cell surface and that regulation of activity is not dependent on alterations in membrane movement.
Differentiation of HL‐60 cells is associated with an increase in the 35‐kDa protein lipocortin I
Authors: William Felicia , Mroczkowski Barbara , Cohen Stanley , Kraft Andrew S.
Published: February 2005

Lipocortin I, a 35‐kDa protein, has been detected in terminally differentiated monocytes and neutrophils. This calcium‐phospholipid binding protein appears to be identical to a 35‐kDa protein that can serve as a substrate for the EGF‐receptor/tryosine kinase. We have used the human myelocytic cell line HL‐60 to explore whether differentiation of hematopoietic cells is associated with changes in the level of lipocortin I. We find that differentiation of HL‐60 cells toward the macrophage lineage by the addition of phorbol esters or vitamin D3 or toward neutrophils with dibutyryl cyclic AMP or dimethyl sulfoxide is accompanied by an increase in the cellular content of lipocortin I. In comparison, treatment of HL‐60 cells with bryostatin 1, a compound that activates protein kinase C but does not differentiate HL‐60 cells, did not effect the level of 35 kDa protein. We have developed a radioimmunoassay to quantitate this protein by using a polyclonal antibody to a synthetic amino terminal peptide of the 35‐kDa protein. This antibody recognizes purified pig lung 35‐kDa protein as well as a single 35‐kDa protein in HL‐60 and A‐431 cells as determined by Western blotting and immune precipitation. Differentiated HL‐60 cells contain 2.6‐fold the amount of 35‐kDa protein found in undifferentiated HL‐60 cells. Our findings that the addition of phorbol esters to HL‐60 cells results in an increase in the mRNA for the 35‐kDa protein and in an increase in the incorporation of 35S‐methionine into the protein suggest that transcriptional activation or increased stability of the mRNA is responsible for the increased rate of synthesis and accumulation of lipocortin I during differentiation of these cells. In the absence of added divalent cations, we have determined that in differentiated HL‐60 cells 79% of lipocortin I protein is located in the cytosol while 21% of the total cellular protein is bound to the particulate fraction. The 35‐kDa protein can be removed from the particulate fraction by incubation with chelators or treatment with phospholipase A2 or phospholipase C. Addition of the calcium ionophore A23187 to intact differentiated HL‐60 cells causes the 35‐kDa protein to associate with the particulate fraction of the cell, suggesting that modulation of intracellular calcium levels may play a role in changing the intracellular location of this protein.
Sulfogalactolipid binding protein SLIP 1: A conserved function for a conserved protein
Authors: Law H. , Itkonnen O. , Lingwood C. A.
Published: February 2005

We have studied the species and tissue expression of the 68kD sulfogalactolipid binding protein SLIP 1, originally detected in the male germ cells of the rat (Lingwood: Can. J. Biochem. Cell Biol., 63:1077–1085, 1985). OUr results show that SLIP 1 has been highly conserved during evolution and is found in the testes of all vertebrates tested. In studies in the rat, we have found that SLIP 1 is, however, tissue restricted, being found only in the brain (also a major site of sulfogalactolipid biosynthesis) in addition to the testis. SLIP 1 was also detected in mammalian oocytes. The SLIP 1 species detected in brain and oocytes retain the sulfogalactolipid‐binding characteristics of rat testicular SLIP 1, indicating that, in addition to immunological features, the glycolipid‐binding function of SLIP 1 is conserved in these tissues.
Receptor‐mediated uptake of a mannose‐6‐phosphate bearing glycoprotein by isolated chicken osteoclasts
Authors: Blair Harry C. , Teitelbaum Steven L. , Schimke Patricia A. , Konsek John D. , Koziol Catherine M. , Schlesinger Paul H.
Published: February 2005

We have recently shown that degradation of bone collagen by osteoclasts occurs via proteolytic enzyme activity that depends on an acidic milieu. Since bone resorption occurs in an extracellular, acidic compartment located at the cell‐matrix attachment site, the osteoclast must deliver the acid collagenolytic enzymes to the cell surface. These observations raise the possibility that the mannose‐6‐phosphate (M‐6‐P) receptor, known to sort acidic proteases in other cells, is involved in trafficking lysosomal enzymes to the plasmalemma of bone resorbing cells. To this end we studied receptor‐mediated uptake, distribution and release, by isolated chicken osteoclasts, of 125l‐hexosaminidase, a M‐6‐P bearing enzyme. We found that at 4°C, the bone‐resorbing polykaryons bind ∼ 10,000 molecules of radioligand/cell with a Kd of 0.7 nM, which is endocytosed by osteoclasts at 37°C by a calcium‐independent process. Furthermore, 125l‐hexosaminidase uptake is unaffected by mannosylated albumin, documenting specificity of the receptor‐mediated event. Release of endocytosed enzyme from the cell is also much more rapid than its degradation, attesting to a pathway of uptake and secretion. By autoradiography, the M‐6‐P bearing ligand is concentrated at the site of osteoclast‐bone attachment. Thus, osteoclasts also have the capacity to deliver M‐6‐P bearing degradative enzymes to their surface at the site of matrix degradation.
Organic cation uptake by a cultured renal epithelium
Authors: McKinney T. Dwight , DeLeon Catherine , Speeg K. Vincent
Published: February 2005

Several organic cations are actively transported by proximal renal tubules by mediated processes across both the apical and basolateral cell membranes. In order to evaluate this transport system in a cultured renal epithelium, uptake of 3H‐tetraethylammonium (TEA) across the apical membrane was measured in LLCPK1 cells, a cell line with several characteristics of proximal tubules. 3H‐TEA progressively entered these cells and reached a near‐steady state by 30 min. Three‐minute uptake was saturable with an apparent Vmax of 1,669 ± 129 fmoles/μg DNA and apparent Km of 34.0 ± 3.4 μM. 3H‐TEA uptake was inhibited by an excess of nonradioactive TEA, other organic cations, sodium azide, and hypothermia. An alkaline external pH was associated with greater 3H‐TEA uptake than an acid pH. However, efflux of 3H‐TEA from cells was not appreciably affected by changes in external pH. Preincubation of cells in acid or alkaline media did not affect uptake. Alteration of cell pH by ammonium chloride addition or removal had little effect on 3H‐TEA uptake. Finally, uptake of 3H‐TEA was not accelerated by preloading cells with an excess of nonradioactive TEA. These results indicate that intact LLCPK1 cells possess a mechanism(s) in their apical membranes for the mediated transport of a prototypic organic cation. The mechanism(s) involved in this transport is uncertain. However, neither organic cation/proton nor organic cation/organic cation exchange appears to be the predominant process.
Loss of low‐affinity serotonin receptors upon storage of human platelets
Authors: Soslau Gerald , Gillen Judy , Parker Janet
Published: February 2005

Platelets actively accumulate virtually all plasma serotonin within their dense granules. As a readily isolated, homogeneous cell type, platelets have served as a model for serotonin uptake into neurological tissue, in addition to defining the role of serotonin in hemostasis. The number of serotonin receptor types on the platelet membrane and the function of these receptors has not been conclusively demonstrated. The presence of different receptor types that may be altered or lost in disease or upon aging (in vitro storage or in vivo) could have significant physiological effects on platelet function. This report demonstrates that at least two receptor types are present on freshly prepared human platelets. However, after 3 to 4 days of storage in autologous plasma, the low‐affinity, high‐capacity serotonin receptor appears to be lost. This phenomenon probably accounts for some of the discrepancies reported in the literature. The high‐affinity receptor present in both freshly isolated and stored platelets binds about 9 × 103 serotonin molecules per platelet. Binding can be completely blocked by imipramine; however, some passive diffusion appears to occur even at the low level of extracellular serotonin concentrations employed in these studies (nanomolar range). The influx of serotonin into platelets appears to be poorly reversible, even in reserpine‐treated cells, where the extravesicular cytoplasmic concentration would be high. The loss of the low‐affinity serotonin receptor type reported in these studies may be directly or indirectly associated with the reduced responsiveness observed in stored platelets.
cAMP‐mediated growth inhibition of a B‐lymphoid precursor cell line Reh is associated with an early transient delay in G2/M, followed by an accumulation of cells in G1
Authors: Blomhoff Heidi Kiil , Blomhoff Rune , Stokke Trond , Davies Catharina DeLange , Brevik Kristin , Smeland Erlend B. , Funderud Steinar , Godal Tore
Published: February 2005

This study was undertaken to gain more insight into the effects of cyclic adenosine monophosphate (cAMP) on cell‐cycle progression in the B‐lymphoid precursor cell line Reh. The adenylate cyclase activator forskolin reduced the proliferation of asynchronously growing Reh cells by 50% after 72 hr culture. Growth inhibition was associated with an accumulation of cells in G1. Furthermore, we demonstrated that forskolin provoked a delay of cells for ∼ 10 hr in G2/M prior to the G1 arrest. Two different methods were applied to elucidate how cells in different phases of the cell cycle were affected by an elevated cAMP level. One method was based on centrifugal elutriation, whereby synchronous cell populations from the different phases of the cell cycle were isolated. By the other method, S‐phase cells were selectively stained by pulsing asynchronously growing cells with bromo‐deoxyuridine (BrdU). The data demonstrate that the position of a cell in the cell cycle is critical in determining how the cell will respond to an elevated cAMP level. Thus cells in G1 at the time forskolin is added are not delayed in G2/M, but they will subsequently accumulate in G1 after 48 hr. Cells given forskolin in G2/M, however, are delayed for 10 hr in G2/M, but they do not accumulate in G1. Cells given forskolin in the S phase are delayed in G2/M as well as arrested in G1. The results suggest that cAMP inhibits growth of the Reh cells by preventing the cells from passing important restriction points located in the G1 and G2 phases of the cell cycle.
Sex hormones modulate the synthesis of basic fibroblast growth factor in human endometrial adenocarcinoma cells: Implications for the neovascularization of normal and neoplastic endometrium
Authors: Presta Marco
Published: February 2005

Basic fibroblast growth factor (bFGF) has been demonstrated to exert an angiogenic activity in vivo. Here, the ability of bFGF to stimulate plasminogen activator (PA) production in bovine capillary endothelial cells was used as an assay for the presence of bFGF‐like molecules in the extracts of the human endometrial adenocarcinoma AN3CA, HEC‐1‐A, and HEC‐1‐B cell lines. The identity of the PA‐stimulating activity with bFGF was confirmed by its high affinity for heparin and by its cross‐reactivity with antibodies to human placental bFGF. Western blot analysis and immunoprecipitation experiments showed that, in the extracts of the three cell lines, these antibodies recognize a protein with an apparent molecular weight of ∼ 17,000 daltons. 17β‐Estradiol stimulates the synthesis of this bFGF‐like molecule in all the endometrial cell lines tested. This stimulation can be abolished by treating the cells with progesterone. These data demonstrate the capacity of sex hormones to regulate bFGF synthesis in tumor endometrial cells, and they suggest that bFGF may play a role in the vascularization of the endometrial adenocarcinoma as well as of the normal endometrium.
L‐ascorbic acid 2‐phosphate stimulates collagen accumulation, cell proliferation, and formation of a three‐dimensional tissuelike substance by skin fibroblasts
Authors: Hata Ryu‐Ichiro , Senoo Haruki
Published: February 2005

Proliferation of human skin fibroblasts was stimulated significantly by the presence of L‐ascorbic acid 2‐phosphate (Asc 2‐P). The presence of Asc 2‐P (0.1–1.0 mM) in the culture medium for 3 weeks enhanced the relative rate of collagen synthesis to total protein synthesis 2‐fold as well as cell growth 4‐fold. Coexistence of L‐azetidine 2‐carboxylic acid (AzC), an inhibitor of collagen synthesis, attenuated both effects of Asc 2‐P in a dose‐dependent manner. Supplementation of the medium with Asc 2‐P also accelerated procollagen processing to collagen and deposition of collagen in the cell layer. Among the acidic glycosaminoglycans (GAG), another major component of extracellular matrix (ECM), deposition of sulfated forms was increased by the additive. Electron microscopic observations showed multilayered, rough endoplasmic reticulum‐rich cells surrounded by dense ECM. These results indicate that Asc 2‐P is useful in culture systems as a long‐acting vitamin C derivative and also that it promotes reorganization of a three‐dimensional tissuelike substance from skin fibroblasts in culture by stimulating collagen accumulation in the fibroblasts.
Characterization of the inhibitory effect of glucocorticoids on the DNA replication of adult rat hepatocytes growing at various cell densities
Authors: Vintermyr Olav Karsten , Døskeland Stein Ove
Published: February 2005

Dexamethasone inhibited the basal and EGF‐stimulated DNA synthesis of adult rat hepatocytes in primary culture. The inhibition was glucocorticoid‐specific: It was shown by dexamethasone and hydrocortisone, but not by progesterone, testosterone, or estradiol; and was counteracted by the glucocorticoid antagonist RU‐38486 in a concentration‐dependent manner. Dexamethasone acted by decreasing the rate of entry into S‐phase (kG1/S), while cell cycle parameters were unaffected. The steroid was able to decrease the kG1/S severalfold even when added more than 20 hr after EGF, half‐maximal effect occurring 11 hr after the addition of dexamethasone. Densily populated areas were much more sensitive to the inhibition by dexamethasone than sparsely populated areas within the same culture dish: A moderate (10 nM) concentration of dexamethasone nearly abolished the DNA synthesis in densily populated areas of hepatocyte cultures with only marginal effect on sparsely populated cells.
Immunodetection and quantitation of the two forms of transforming growth factor‐beta (TGF‐β1 and TGF‐β2) secreted by cells in culture
Authors: Danielpour David , Dart Linda L. , Flanders Kathleen C. , Roberts Anita B. , Sporn Michael B.
Published: February 2005

Transforming growth factor beta (TGF‐β), a potent modulator of cell growth, differentiation, and the expression of extracellular matrix components in a variety of cell types, exists as two distinct homodimers (TGF‐β1 and TGF‐β2), sharing 71% sequence homology. Radioreceptor and previously described radioimmunological assays using rabbit antibodies have not been able to distinguish between these two forms. We have developed antisera in turkeys against native TGF‐β1 and TGF‐β2, each of which specifically blocks both the receptor binding and biological activity of each of these peptides. With these immunological reagents we describe sensitive and specific immunological assays for TGF‐β1 and TGF‐β2 in complex biological fluids. Using these assays we show that both TGF‐β1 and TGF‐β2 are secreted by a variety of cultured cells, but that some cells secrete predominantly either TGF‐β1 or TGF‐β2 while others secrete both peptides in nearly equal amounts. Our results demonstrate that the expression of each of the two forms of TGF‐β is independently regulated.
M2 subunit of ribonucleotide reductase is a target of cyclic AMP‐dependent protein kinase
Authors: Albert Daniel A. , Nodzenski Edwardine
Published: February 2005

Cyclic AMP arrests T lymphocytes in the G1 phase of the cell cycle, and prolonged exposure results in cytolysis. Both of these effects require cyclic AMP‐dependent protein kinase. We recently observed that some S49 mouse T lymphoma cell lines selected for hydroxyurea resistance were not arrested in G1 by cyclic AMP. Further analysis revealed that these cell lines were cyclic AMP‐dependent protein kinase deficient, and conversely, other cyclic AMP‐dependent protein kinase deficient cell lines not selected for hydroxyurea resistance were two‐ to threefold more hydroxyurea resistant. However, hydroxyurea is a specific inhibitor of ribonucleotide reductase and does not inhibit this kinase. We subsequently showed that cyclic AMP‐dependent protein kinase will phosphorylate the M2 but not the M1 subunit of ribonucleotide reductase in vitro, and this phosphorylation will diminish CDP reductase activity. In vivo phosphorylation of M2 occurred under conditions similar to those that generate cell cycle arrest. We conclude that the M2 subunit of ribonucleotide reductase can be a target of cyclic AMP‐dependent protein kinase. The phosphorylated enzyme has diminished activity, and this may play a role in cyclic AMP‐induced lymphocyte cell cycle arrest.
Endocytosis of follicle‐stimulating hormone by ovarian granulosa cells: Analysis of hormone processing and receptor dynamics
Authors: Sanford Jack C. , Batten Bruce E.
Published: February 2005

Suspensions of freshly isolated rat granulosa cells were used to study endocytosis and processing of radioiodinated ovine follicle‐stimulating hormone (l‐oFSH) and to analyze the dynamics of its receptor. Ovine FSH was iodinated to a specific activity of 26 μCi/μg as determined by radioreceptor self‐displacement assays with maximum specific binding to excess membrane receptors of 46%. Radiolabeled oFSH was judged biologically equivalent to the unlabeled hormone since l‐oFSH shows saturation‐binding kinetics and stimulates steroidogenesis in a similar dose‐related manner to unlabeled oFSH. Experiments designed to study the extent and time course of degradation involved continuous exposure of isolated granulosa cells to l‐oFSH. Saturation of membrane receptors was achieved within 1.5 h of incubation, and internalization of FSH occurred in a linear manner for up to 6 h. The rate of internalization was equivalent to 2,780 FSH molecules/cell/h. Degradation of FSH became apparent after 6 h of incubation and increased to 86% of total cellular‐associated radioactivity at 22 h. FSH degradation was inhibited by 100 μM chloroquine or 0.45 mM leupeptin. The measurement of cell surface l‐oFSH binding in the combined presence of 100 μM chloroquine and 0.5 mM cycloheximide was unchanged for up to 22 h of incubation. This and other receptor binding data suggest that there is no reutilization of FSH receptors. Scatchard analyses of 4°C binding assays on intact cells indicated that a two‐site model best fit the data with association constants of K11 = 1.44 (±.42) × 1010 and K12 = 4.35 (±.91) × 108. Receptor binding and activation studies for progesterone production yielded ED50S of 270 pM and 7.7 pM, respectively, and also indicated that 20% receptor occupancy is sufficient to stimulate maximal progesterone production. We conclude that after the initial binding event, FSH is endocytosed very slowly and is subsequently shuttled to the lysosomal compartment for degradation. The retarded rate of endocytosis may relate to novel pathways of hormone processing.
In vitro effects of endotoxin on bovine and sheep lung microvascular and pulmonary artery endothelial cells
Authors: Meyrick Barbara , Hoover Richard , Jones Margaret R. , Berry Leonard C. , Brigham Kenneth L.
Published: February 2005

A single infusion of Escherichia coli endotoxin into sheep results in structural evidence of pulmonary endothelial injury, increases in both prostacyclin and prostaglandin E2 (PGE2) in lung lymph, and an increase in pulmonary micro‐vascular permeability. Endotoxin‐induced lung endothelial damage can also be induced in vitro, but to date these studies have utilized endothelium from large pulmonary vessels. In the present study, we have grown endothelial cells from peripheral lung vessels of cows and sheep and exposed these microvascular endothelial cells to endotoxin. Controls included lung microvascular endothelium without endotoxin and endothelial cells from bovine and sheep main pulmonary artery with and without addition of endotoxin. We found that endotoxin caused significant increases in release of prostacyclin and PGE2 from both bovine and sheep lung microvascular and pulmonary artery endothelium. Normal bovine and sheep pulmonary artery and bovine lung microvascular endothelium released greater levels of prostacyclin than PGE2 (ng/ng); release of PGE2 from the microvascular cells was greater than from the pulmonary artery endothelium in both species. Exposure of endothelial cells from cow and sheep main pulmonary artery to endotoxin results in endothelial cell retraction and pyknosis, a loss of barrier function, increased release of prostacyclin and PGE2 and eventual cell lysis. In lung microvascular cells, the increases in prostanoids were accompanied by changes in cell shape but occurred in the absence of either detectable alterations in barrier function or cytolysis. Thus, while endotoxin causes alterations to endothelial cells from both large and small pulmonary vessels, the effects are not identical suggesting site specific phenotypic expression of endothelial cells even within a single vessel. To determine whether the response of either the large or small pulmonary vessel endothelial cells in culture mimics most closely the in vivo response of the lung to endotoxin requires further study.
Combinative ligand‐receptor interactions: Effects of CAMP, epinephrine, and met‐enkephalin on RAW264 macrophage morphology, spreading, adherence, and microfilaments
Authors: Petty Howard R. , Martin Susan M.
Published: February 2005

Cell surface ligand‐receptor interactions play a central role in the regulation and expression of macrophage function. Included among these macrophage membrane receptors are the β‐adrenergic and opioid receptors. We studied the abilities of epinephrine, met‐enkephalin, forskolin, and adenosine 3′:5′ cyclic monophosphate (cAMP) analogues to affect macrophage morphology, spreading, and adherence. Cell spreading was quantitated by measuring the perimeters of adherent cell images recorded by videomicroscopy. Epinephrine induced a dose‐dependent decrease in macrophage spreading; at 10‐5 M epinephrine the mean perimeter was 10.4 ± 0.3μm in comparison to 15.0 ± 1.0 μm for controls. The inhibition of spreading can be blocked by the antagonist propranolol. On the other hand, met‐enkephalin induced a dose‐dependent increase in macrophage spreading, with a perimeter of 18.5 ± 1.0 μm at 10‐8 M. Since catecholamines and opioids are simultaneously released from chromaffin cells of the adrenal, we examined the combinative effects due to treatment with both ligands. When macrophages were exposed to 10‐5 M epinephrine and 10‐8 M met‐enkephalin, cell morphology and spreading were indistinguishable from that due to 10‐5 M epinephrine alone. The epinephrine dose‐response curve in the presence of 10‐8 M met‐enkephalin, was similar to that of epinephrine alone. The β‐adrenergic receptor is apparently capable of diminishing or abrogating the opioid receptor signal(s). These combinative and epinephrine‐mediated effects may be at least partially accounted for by the action of cAMP. Forskolin and the cAMP analogues N6 –2′‐O‐dibutyryladenosine 3′:5′ cyclic monophosphate (dbcAMP) and 8‐bromoadenosine 3′:5′ cyclic monophosphate (Br‐cAMP) affected cell morphology and spreading in the same fashion as epinephrine. These differences in morphology and spreading behavior were accompanied by changes in the distribution of F‐actin, as judged by phalladicin staining and fluorescence microscopy. We suggest that cAMP and microfilaments play important roles in receptor‐mediated neuroregulation of macrophage function.
Ionic dependence of the extracellular ATP‐induced permeabilization of transformed mouse fibroblasts: Role of plasma membrane activities that regulate cell volume
Authors: Weisman Gary A. , De Barun K. , Pritchard Robert S.
Published: February 2005

Extracellular ATP rendered the plasma membrane of transformed mouse fibro‐blasts permeable to normally impermeant molecules. This permeability change was prevented by increasing the ionic strength of the isotonic medium with NaCl. Conversely, the cells exhibited increased sensitivity to ATP when the NaCl concentration was decreased below isotonicity, when the KCl concentration was increased above 5 mM while maintaining isotonicity, and when the pH of the medium was raised above 7.0. These conditions as well as the addition of ATP itself caused cell swelling. However, the effect of ATP was independent of cell volume and dependent upon the ionic strength and not the osmolarity of the medium since (1) addition of sucrose to isotonic medium did not prevent permeabilization although media made hypertonic with either sucrose or NaCl caused a decrease in cell volume; and (2) addition of sucrose or NaCl to hypotonic media caused a decrease in cell volume, but only NaCl addition decreased the response to ATP. Conditions that have been shown to inhibit plasma membrane proteins that play a reciprocal role in cell volume regulation had reciprocal effects on the permeabilization process, even though the effect of ATP was independent of cell volume. For example, inhibition of the Na+, K+‐ATPase by ouabain increased sensitivity of cells to ATP while conditions which inhibit Na+, K+, Cl−‐cotransporter activity, such as treatment of the cells with the diuretics furosemide or bumetanide or replacement of sodium chloride in the medium with sodium nitrate or thiocyanate, inhibited permeabilization. The furosemide concentration that inhibited permeabilization was greater than the concentration that inhibited Na+, K+, Cl−‐cotransporter‐mediated 86Rb+ (K+) uptake, suggesting that the effect of furosemide on the permeabilization process may not be specific for the Na+, K+, Cl−‐cotransporter.
Electric fields stimulate DNA synthesis of mouse osteoblast‐like cells (MC3T3‐E1) by a mechanism involving calcium ions
Authors: Ozawa Hiroyuki , Abe Etsuko , Shibasaki Yoshinobu , Fukuhara Tatsuo , Suda Tatsuo
Published: February 2005

A cell culture system was designed to study the effect of an electric field on bone cells. This system subjected cultured bone cells to a series of high‐voltage pulses at varying amplitudes of electric field without changing the ion concentrations of the culture medium. When mouse osteoblast‐like cells (MC3T3‐E1) were stimulated for 5 min to 20 hr by an electric field (3.19 kV/m, 3 msec, 10 Hz pulses), DNA synthesis was greatly increased. The stimulation occurred only during the growth phase. The electric field also increased incorporation of 45Ca into the cells, but it did not stimulate cAMP production. Adding calcium ionophore A23187 stimulated both 45Ca uptake and DNA synthesis, but dibutyryl cAMP did not stimulate either of them. These results suggest that the electric field stimulates the DNA synthesis of growing osteoblasts by a mechanism involving calcium ions.
Growth of normal human keratinocytes and fibroblasts in serum‐free medium is stimulated by acidic and basic fibroblast growth factor
Authors: Shipley Gary D. , Keeble Winifred W. , Hendrickson Jill E. , Coffey Robert J. , Pittelkow Mark R.
Published: February 2005

Keratinocytes and fibroblasts isolated from human neonatal foreskin can be plated and grown through multiple rounds of division in vitro under defined serum‐free conditions. We utilized these growth conditions to examine the mitogenic potential of acidic and basic fibroblast growth factor (aFGF and bFGF) on these cells. Our results demonstrate that both aFGF and bFGF can stimulate the proliferation of keratinocytes and fibroblasts. aFGF is a more potent mitogen than bFGF for keratinocytes. In contrast, bFGF appears to be more potent than aFGF in stimulating the growth of fibroblast cultures. Heparin sulfate (10 μg/ml) dramatically inhibited the ability of bFGF to stimulate the proliferation of keratinocytes. In comparison, heparin slightly inhibited the stimulatory effect of aFGF and had no effect on epidermal growth factor (EGF) stimulation in keratinocyte cultures. In fibroblast cultures the addition of heparin enhanced the mitogenic effect of aFGF, had a minimal stimulatory effect on the mitogenic activity of bFGF, and had no effect on EGF‐stimulated growth. Our results demonstrate that the proliferation in vitro of two normal cell types found in the skin can be influenced by aFGF and bFGF and demonstrate cell‐type specific differences in the responsiveness of fibroblasts and keratinocytes to these growth factors and heparin.
Nonterminally differentiated cells express decreased growth factor responsiveness
Authors: Hoerl Bryan J. , Scott Robert E.
Published: February 2005

In 3T3 T mesenchymal stem cells, at least four types of biological states exist that can mediate the control of cell differentiation and/or proliferation. These include the predifferentiation growth arrest state, the nonterminal differentiation state, the terminal differentiation state, and a growth arrest state induced by growth factor/serum deficiency. The current studies were performed to investigate the relative mitogenic responsiveness of cells at these four states and specifically to determine if nonterminally differentiated cells show decreased responsiveness to specific mitogens. Twenty‐five different serum, plasma, and growth factor combinations were evaluated. The results show that undifferentiated, growth‐arrested cells are highly responsive to numerous mitogens and that by definition terminally differentiated cells are not responsive to any mitogens. In contrast, nonterminally differentiated cells demonstrate a unique pattern of mitogenic responsiveness. Whereas nonterminally differentiated cells can be stimulated to proliferate by high concentrations of serum or plasma supplemented with growth factors, they cannot be stimulated to proliferate by combinations of multiple purified growth factors. These results suggest that the process of nonterminal differentiation is associated with a significant change in factors/cofactors required to stimulate cell proliferation and that these factors/cofactors are present in plasma.
Effects of n‐alcohols on junctional coupling and amylase secretion of pancreatic acinar cells
Authors: Chanson Marc , Bruzzone Roberto , Bosco Domenico , Meda Paolo
Published: February 2005

We have tested the effects of alcohols differing by their alkyl chain length on the membrane channels and amylase secretion of rat pancreatic acinar cells. In intact acini, alcohols with a chain of seven, eight, or nine carbons (C‐7, C‐8, and C‐9) induced dye uncoupling and increased basal amylase release. These effects were readily reversible after alcohol removal. By contrast, an alcohol with a chain of 15 carbons (C‐15) and several alcohols with chains of fewer than six carbons (C‐2, C‐4, and C‐6) did not uncouple acinar cells and had no effects on amylase secretion. Neither did alkanes and oxidized derivatives of C‐7 and C‐8 alcohols did not affect dye coupling. Double patch‐clamp experiments on pairs of acinar cells, under conditions of strong cytosolic Ca2+ and pH buffering, showed that C‐7, C‐8, and C‐9 alcohols blocked completely and reversibly the electrical conductance of junctional channels. Furthermore, studies of single voltage‐clamped acinar cells revealed that the uncoupling alcohols did not affect the resting nonjunctional membrane conductances. Thus the alcohols that did not affect acinar cell coupling did not affect amylase secretion, whereas the alcohols that caused uncoupling increased secretion. The latter effect was not mediated by changes in the conductance of nonjunctional membrane, cytosolic Ca2+, and pH and, as revealed by an immunological hemolytic plaque assay for amylase, had a time course consistent with the rapid (within 1 min) inhibition of coupling. These data provide new support for the view that the regulation of cell‐to‐cell communications is correlated with that of digestive enzyme secretion.
Differential effects of follicle‐stimulating hormone, insulin, and insulin‐like growth factor I on hexose uptake and lactate production by rat Sertoli cells
Authors: Oonk Ria B. , Jansen Ruud , Grootegoed J. Anton
Published: February 2005

The stimulatory effects of follicle‐stimulating hormone (FSH), insulin, and insulin‐like growth factor I (IGF‐I) on lactate production and hexose uptake by Sertoli cells from immature rats were studied. The time‐courses and the maximal stimulatory effects of FSH, insulin, and IGF‐I on lactate production were virtually identical. When Sertoli cells were incubated in the presence of FSH in combination with insulin or IGF‐I (submaximal doses), additive but no pronounced synergistic effects were observed. The stimulatory effects of FSH and insulin were not dependent on the presence of extracellular calcium. 2‐Deoxy‐D‐glucose (2‐DOG), an analogue of D‐glucose, was used to investigate the hexose transport system of Sertoli cells. Uptake of 2‐DOG was linear in time and virtually all of the intracellular 2‐DOG was phosphorylated up to 30 min of incubation; 2‐DOG uptake was inhibited by c ytochalasin B, but not by cytochalasin E. D‐glucose, but not D‐galactose, appeared to be an effective competitor of 2‐DOG uptake. The Km of 2‐DOG uptake was not influenced by FSH, insulin, and IGF‐I. FSH had no effect on the Vmax of 2‐DOG uptake, whereas insulin and IGF‐I caused a 30% stimulation of the Vmax. It is concluded that FSH, insulin, and IGF‐I stimulate lactate production by cultured Sertoli cells, but that only insulin and IGF‐I stimulate hexose transport. The insulin‐like effect of FSH or Sertoli cells may principally involve stimulation of glycolytic enzyme activities.
Specific interactions of histone H1 and a 45 kilodalton nuclear protein with a putative matrix attachment site in the distal promoter region of a cell cycle‐regulated human histone gene
Authors: Pauli U. , Chiu J. F. , Ditullio P. , Kroeger P. , Shalhoub V. , Rowe T. , Stein G. , Stein J.
Published: February 2005

Protein‐DNA interactions within the promoter of a cell cycle‐regulated human H4 histone gene were examined by binding of 5′‐end‐labeled DNA segments to Western blots of nuclear protein fractions. Specific protein interactions were observed with DNA segments located between −500 bp and −1,070 bp upstream of the ATG initiation codon and included a histone H1 binding segment flanked on both sides by binding sites for a 45 kD nuclear protein. This region of the gene contains a DNase I‐sensitive site in the center (−720 to −820 bp), and sequence analysis revealed the presence of scaffold attachment sequences in the two flanking segments. Topoisomerase II consensus sequences and in vitro topoisomerase II cleavage sites were also detected in the two flanking segments. Our results suggest that the 45 kd nuclear protein may preferentially interact with these two segments of the H4 histone gene to mediate association with the nuclear matrix. The presence of negative regulatory elements in this putative matrix attachment region provides a basis for the speculation that such nuclear proteins are associated with alterations in gene‐matrix interaction that are functionally related to gene expression.
Epidermal growth factor binding, stimulation of phosphorylation, and inhibition of gluconeogenesis in rat proximal tubule
Authors: Harris Raymond C. , Daniel Thomas O.
Published: February 2005

Epidermal growth factor and insulin share many biological activities, including stimulation of cell proliferation, ion flux, glycolysis, fatty acid and glycogen synthesis, and activation of receptor‐linked tyrosine kinase activity. In the kidney, insulin has been shown to regulate transport processes and inhibit gluconeogenesis in the proximal tubule. Since the kidney represents a major source of EGF, the present studies investigated whether proximal tubule contained EGF receptors, whether EGF receptors were localized to apical or basolateral membranes, and whether EGF receptor activation participated in the regulation of an important proximal tubule function, gluconeogenesis.

Specific EGF receptors were demonstrated in the basolateral membrane of proximal tubule. Following incubation with 125l EGF, basolateral membranes demonstrated equilibrium binding at 4°C and 23°C. There was 78 ± 2% specific binding (n = 13). The dissociation constant (Kd) was 1.5 × 1 0−9 M and maximal binding was 44 fmol/mg protein. There was ninefold more specific binding to proximal tubule basolateral membrane than to brush border membrane. In basolateral, but not brush border membranes, EGF induced phosphorylation of the tyrosine residues of intrinsic membrane proteins, including a 170 kDa protein, corresponding to the EGF receptor.

In the presence of the gluconeogenic substrates, alanine, lactate, and succinate, proximal tubule suspensions synthesized glucose. EGF inhibited glucose production in a concentration‐dependent manner over a concentration range of 3 × 10−11 to 3 × 10−9 M. In addition, EGF inhibited angiotensin ll‐stimulated glucose production in the proximal tubule suspensions. EGF did not significantly increase net glucose metabolism nor decrease cellular ATP concentrations.

Therefore, these studies demonstrated that rat proximal tubule contained specific receptors for EGF that were localized to the basolateral membrane and linked to tyrosine kinase activity. EGF significantly inhibited proximal tubule glucose production without significantly increasing net glucose consumption.
Second messenger function of phosphatidic acid in platelet activation
Authors: Kroll Michael H. , Zavoico George B. , Schafer Andrew I.
Published: February 2005

Phosphatidic acid (PA) is synthesized as the result of the receptor‐mediated response of platelets to physiologic agonists. The role of PA in platelet signal transduction, however, is largely unknown. We have examined the responses of platelets to 1‐stearoyl‐2‐arachidonoyl phosphatidic acid (SAPA), the predominant molecular species of human platelet PA. SAPA alone causes platelet aggregation, and pretreatment of platelets with SAPA markedly enhances thrombin‐induced aggregation and secretion. Addition of SAPA to intact human platelets causes rapid breakdown of phosphatidylinositol‐4,5‐bisphosphate (PIP2) and the generation of diacylglycerol and endogenous PA. These reactions are associated with mobilization of intracellular calcium and activation of protein kinase C. SAPA also stimulates the release of endogenous arachidonic acid and its conversion to thromboxane A2. Furthermore, platelet activation by SAPA is blocked by indomethacin, indicating that the actions of SAPA are mediated by cyclooxygenase products. These findings suggest that SAPA may play an important role as an endogenous positive feedback signal to amplify receptor‐mediated activation of PIP2‐specific phospholipase C in human platelets.
Pulmonary alveolar macrophages express a polyamine transport system
Authors: Saunders Nicholas A. , Ilett Kenneth F. , Minchin Rodney F.
Published: February 2005

Polyamine transport is an important mechanism by which cells regulate their intracellular polyamine content. It is well established that the lung has a high capacity for polyamine transport, and recently the polyamine putrescine has been shown to be selectively accumulated into the type II pneumocyte of rabbit lung slices (Saunders et al.:Lab. Invest., 95:380–386, 1988). In addition, it has been suggested that there may be more than one polyamine transport system in lung tissue (Byers et al.:Am. J. Physiol., 252:C663‐C669, 1987). In the present study, we have examined whether there are differences in the distribution of putrescine and spermidine uptake activities in isolated rabbit lung cells. We report that pulmonary alveolar macrophages have a greater rate of uptake of both putrescine and spermidine than the total lung cell population. Kinetic analysis of the polyamine uptake system present in macrophages showed putrescine uptake consisted of a saturable (Km = 2.1 μM) and nonsaturable component whilst spermidine uptake consisted of both a high‐ and a low‐capacity saturable component (Km = 0.16 μM and 1.97 μM, respectively). The rate of polyamine transport was similar to those reported for many proliferative or tumor cell‐lines and appeans to be greater than any other major lung cell type. Inhibition studies of the transport of polyamines into pulmonary alveolar macrophages suggested that the uptake of both putrescine and spermidine was mediated by the same system, which could not be described by simple Michaelis‐Menten kinetics. The transport appears to be reversible due to significant efflux. This is the first study to describe the presence of multiple polyamine transport systems in pulmonary alveolar macrophages.