bluuebunny/crossref_metadata_2025 huggingface dataset:
Calcium is essential in the fusion of mouse alveolar macrophages induced by 1α, 25‐dihydroxyvitamin D3
Authors: Jin Cheng He , Segawa Akishisa , Miyaura Chisato , Tanaka Hirofumi , Abe Ersuko , Suda Tatsuo
Published: February 2005

We have reported that the active form of vitamin D3, 1α, 25‐dihydroxyvitamin D3 [1α, 25(OH)2D3], directly induces activation and fusion of mouse alveolar macrophages (Abe et al., 1983, 1984). The activated state appeared to be a prerequisite to the fusion of macrophages. Macrophages began to fuse 36 hr after adding 1α, 25(OH)2D3; the fusion rate attained a maximum of 70–80% at 72 hr. During the course of further investigating the mechanisms of fusion induced by the vitamin, we found that the calcium ion is closely involved in the fusion process of macrophages induced by 1α, 25(OH)2D3. When alveolar macrophages were cultured with 1α, 25(OH)2D3 in medium with graded concentrations (0.13–1.85 mM) of calcium, the fusion rate went down in parallel with the decrease of medium calcium. Neither calcium ionophore A23187 nor 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) induced fusion of freshly isolated macrophages, but the two compounds greatly promoted fusion of the macrophages pretreated for 18 hr with 1α, 25(OH)2D3. The vitamin effect for the first 18 hr was similar, irrespective of the medium calcium concentration. In contrast, millimolar amounts of calcium were essential in the subsequent period of incubation(18–72 hr) for inducing fusion. The activation of macrophages measured by the induction of cytotoxicity and the enhancement of glucose consumption by 1α,25(OH)2D3 occurred similarly, irrespective of the medium calcium concentration. These results clearly indicate that the fusion process of alveolar macrophages induced by 1α, 25(OH)2D3 can be divided into two phases: (1) the calcium‐independent priming phase (0–18 hr) and (2) the calcium‐dependent progression phase (18–72 hr). 1α,25(OH)2D3 is necessary only in the priming phase; A23187 and TPA can be substituted for 1α,25(OH)2D3 in the progression phase.
Comparison of the biological actions of TGF beta‐1 and TGF beta‐2: Differential activity in endothelial cells
Authors: Jennings John C. , Mohan Subburaman , Linkhart Thomas A. , Widstrom Richard , Baylink David J.
Published: February 2005

Beta transforming growth factor (TGF beta) has multiple in vitro biological effects including stimulation or inhibition of proliferation of specific cell types. A second major form of TGF beta, TGF beta‐2, has recently been isolated from porcine platelets, from bovine bone matrix, and from several other sources. The two forms of TGF beta are biologically equipotent with the exception that TGF beta‐2 was much less active than TGF beta‐1 for inhibition of proliferation of a rat pleuripotent hematopoietic stem cell line. During the purification of beta TGF from bone, we obtained two fraction pools that differed in their ability to inhibit 3H‐thymidine incorporation into aortic endothelial cells (AEC). We therefore compared highly purified TGF beta‐1 and TGF beta‐2 isolated from porcine platelets for inhibition of DNA synthesis in mink lung epithelial cells (MvlLu), and in AEC, and for stimulation of 3H‐thymidine incorporation in calvarial bone cells (CBC) in 3 experiments. TGF beta‐1 and TGF beta‐2 inhibited cell proliferation in MvlLu with no significant differences in the ED50 (31± 8pg/ml vs 23± 7). TGF beta‐2 was much less potent than TGF beta‐1 in inhibiting DNA synthesis in AEC (6310 ± 985 pg/ml vs 101 ± 34). The reduced specific activity of TGF beta‐2 was also observed in adrenal capillary endothelial cells. Both beta‐1 and beta‐2 stimulated proliferation of CBC (ED50 26 ± 2 pg/ml vs 10 ± 4). We also examined the specificity of the MvlLu and AEC inhibition assays. Epidermal growth factor (EGF), platelet derived growth factor (PDGF), acidic and basic fibroblast growth factor (FGF), skeletal growth factor (SGF)/insulin‐like growth factor‐II (IGF‐II), and insulin‐like growth factor‐I (IGF‐I) did not inhibit DNA synthesis in either assay system. However, when the growth factors were added to maximal inhibiting concentrations of TGF beta‐1, both acidic and basic FGF significantly reduced TGF beta‐1 inhibition in AEC. We conclude that (1) inhibition of DNA synthesis in endothelial cells is relatively specific for TGF beta‐1, (2) inhibition of DNA synthesis in MvlLu is a sensitive and specific assay for generic TGF beta activity but does not distinguish beta‐1 from beta‐2, (3) the relative inhibition of DNA synthesis in MvlLu and AEC may provide a means to quantitatively estimate TGF beta‐1 and TGF beta‐2, and (4) both TGF beta‐1 ad TGF beta‐2 are potent mitogens for chicken embryonic calvarial bone cells.
Role of polyamines in the stimulation of synthesis and secretion of plasminogen activator from bovine aortic endothelial cells
Authors: Kuo Be‐Sheng , Korner Gil , Dryjski Maciej , Bjornsson Thorir D.
Published: February 2005

The effects of the polyamines putrescine (PUT), spermidine (SPD), and spermine (SPM) on the secretion of plasminogen activator (PA) and plasminogen activator inhibitor (PAI) were evaluated using cultured bovine aortic endothelial cells. All three polyamines enhanced PA secretion in a time‐ and dose‐dependent manner, with a potency rank order of SPM > SPD > PUT. The PA stimulation required both RNA and protein synthesis, as evidenced by inhibition of polyamine‐induced PA secretion by actinomycin D and cycloheximide. The inhibitors of polyamine biosynthesis methylglyoxal bis‐(guanylhydrazone) (MGBG) and dl‐(difluoromethyl) ornithine (DFMO) alone did not affect basal or polyamine‐induced PA secretion, with the exception that MGBG reduced the effect of PUT. Polyamine‐treated cells enhanced secretions of both tissue‐type and urokinase‐type PA. The results of the present study suggest that polyamines may play a role in the regulation of PA synthesis and secretion and that this function can be modified under pathophysiological conditions affecting cellular and tissue levels of polyamines.
Cyclic AMP increasing agents rapidly stimulate vimentin phosphorylation in quiescent cultures of Swiss 3T3 cells
Authors: Escribano Josefa , Rozengurt Enrique
Published: February 2005

The results presented here demonstrate that an elevation in the cellular levels of cyclic AMP (cAMP) increases the phosphorylation of an Mr = 58,000 cellular protein in quiescent cultures of Swiss 3T3 cells. The enhancement of 32Pi incorporation into the Mr = 58,000 cellular protein was detected as early as 1 min and reached a maximum after 20 min of treatment. The role of cAMP in the phosphorylation of Mr = 58,000 protein is substantiated by the following lines of evidence: (a) a variety of agents that cause cAMP accumulation in 3T3 cells, including cholera toxin, 5′‐N‐ethylcarboxamideadenosine (NECA), PGE1, and 3‐isobutyl‐1‐methyl‐xanthine (IBMX) increased the phosphorylation of the same Mr 58,000 cellular protein as demonstrated by peptide mapping; (b) inhibitors of cyclic nucleotide phosphodiesterase potentiated the ability of low concentrations of the adenylate cyclase activators NECA, PGE1, and forskolin to increase Mr 58,000 phosphorylation; and (c) permeable derivatives of cAMP such as 8BrcAMP were also effective and specific in promoting Mr 58,000 phosphorylation. Detergent extraction, immunoblotting, and immunoprecipitation identified the Mr = 58,000 phosphoprotein as vimentin, the main protein subunit of the intermediate filaments of mesenchymal cells including Swiss 3T3 cells. Studies with intact 3T3 cells revealed that an increase in the intracellular level of cAMP induced a marked redistribution and collapse of the intermediate filaments. These results raise the possibility that an intact intermediate filament network may restrict the reinitiation of DNA synthesis.
Specific binding of basic fibroblast growth factor to basement membrane‐like structures and to purified heparan sulfate proteoglycan of the EHS tumor
Authors: Vigny M. , Ollier‐Hartmann M. P. , Lavigne M. , Fayein N. , Jeanny J. C. , Laurent M. , Courtois Y.
Published: February 2005

The binding of iodinated basic fibroblast growth factor (bFGF) to low‐density heparan sulfate proteoglycan purified from the Engelbreth Holm Swarm (EHS) sarcoma was investigated using different techniques. The tumor clearly contained bFGF, the level being comparable to that found in other tissues such as human or bovine brain. 125I bFGF strongly bound to the basement membrane‐like matrix of EHS frozen sections as revealed by autoradiography. Iodinated bFGF bound to purified heparan sulfate proteoglycan but not to laminin or collagen type IV, three components isolated from the same tumor. In contrast, acidic fibroblast growth factor (aFGF) displayed negligible binding to heparan sulfate proteoglycan. Binding of bFGF to frozen sections and to purified proteoglycan could be strongly inhibited by heparin and was displaced by an excess of unlabeled factor and completely suppressed after heparitinase and heparinase treatments. Binding was a function of the salt concentration and was abolished at 0.6 M NaCl. Scatchard analysis indicated the affinity site had a Kd of about 30 nM, a value 10–15 higher than that recently reported by Moscatelli (J. Cell. Physiol., 131:123–130, 1987) in the case of the low‐affinity binding sites present on the surface of baby hamster kidney (BHK) cells.
Tumor‐promoting phorbol esters stimulate the proliferation of interleukin‐3 dependent cells
Authors: Hogans Beth B. , Spivak Jerry L.
Published: February 2005

To determine whether activation of protein kinase C is involved in the proliferation of interleukin‐3 (IL‐3) ‐dependent cells, we examined the effect of tumor‐promoting phorbol esters on the in vitro proliferation of the IL‐3‐dependent cell lines FD and DA‐1. The viability of FD and DA‐1 cells cultured for 24 hours in 100 nM phorbol myristate acetate (PMA) and 10% FCS was similar to that of cells cultured in 25% WEHI‐3 conditioned medium as a source of IL‐3, and 10% FCS. FD cells failed to proliferate in concentrations of FCS of up to 50%, while DA‐1 cell proliferation was not markedly influenced by FCS. By contrast, PMA promoted the proliferation of FD and DA‐1 cells in the absence of FCS and enhanced their proliferation in the presence of 10% FCS, 60‐ and 20‐fold, respectively. Stimulation of proliferation was achieved with as little as 10 nM PMA and was maximal at 100 nM PMA. Low concentrations (0.05‐0.1%) of WEHI‐3 CM promoted the proliferative response of FD and DA‐1 cells to PMA, but at concentrations of WEHI‐3 CM greater than 0.8%, no further increment in proliferation was obtained with PMA. As little as 1/2 hour of exposure to phorbol esters was sufficient to cause translocation of protein kinase C from the cytosol to the membranes of DA‐1 cells, and 1 hour of exposure to phorbol esters was sufficient to stimulate DNA synthesis. A protein kinase C inhibitor, H‐7, at a concentration of 10 μM inhibited phorbol ester‐induced stimulation of DA‐1 cell proliferation. When DA‐1 cells were exposed to the calcium ionophore A23187 in addition to both a phorbol ester and IL‐3, their proliferation was enhanced over that stimulated by only the phorbol ester and IL‐3. The data indicate that stimulation of proliferation of IL‐3‐dependent cells involves the activation of protein kinase C.
Direct evidence of intercellular sharing of glutathione via metabolic cooperation
Authors: Kavanagh Terrance J. , Martin George M. , Livesey John C. , Rabinovitch Peter S.
Published: February 2005

Intracellular glutathione (GSH) content and cell density are known to be two important determinants of cell sensitivity to free radicals and radiation. We have investigated intercellular sharing of GSH via metabolic cooperation (MC) by measuring the GSH content of Chinese hamster V79 cells under conditions that varied MC among cells. GSH was measured by flow cytometry with monochlorobimane, which becomes fluorescent after conjugation to GSH by GSH‐S‐transferase. High‐performance liquid chromatography was used to confirm the accuracy of GSH measurements by flow cytometry. Several lines of evidence indicate sharing of GSH or its precursor γ‐glutamylcysteine via MC. These include a cell density‐dependent heterogeneity in GSH content, reconstitution of GSH in GSH‐depleted cells by coculture with nondepleted cells (except when the depleted cells were MC deficient), and decreased equilibration of GSH among GSH‐depleted cells and nondepleted cells when an inhibitor of MC (phorbol myristate acetate) was present. The equilibration of GSH among GSH‐depleted cells and nondepleted cells in coculture was not inhibitable by acivicin, suggesting that this form of intercellular sharing of GSH does not rely on γ‐glutamyltransferase‐mediated extracellular transport of GSH.
Effects of glucocorticoids on Na+/H+ exchange and growth in cultured vascular smooth muscle cells
Authors: Berk Bradford C. , Vallega Gino , Griendling Kathy K. , Gordon John B. , Cragoe Edward J. , Canessa Mitzy , Alexander R. Wayne
Published: February 2005

We have examined the effects of hydrocortisone on growth and Na+/H+ exchange in cultured rat aortic vascular smooth muscle cells (VSMC). Hydrocortisone (2 μM) treatment of growth‐arrested VSMC significantly decreased VSMC growth in response to 10% calf serum assayed by 3H‐thymidine incorporation and cell number at confluence. This effect was associated with the appearance of an altered cell phenotype characterized by large, flat VSMC that did not form typical “hillocks.” Na+/H+ exchange was also altered in hydrocortisone‐treated cells assayed by dimethylamiloride‐sensitive 22Na+ influx into acid‐loaded cells or by intracellular pH (pHi) change using the fluorescent dye BCECF. Resting pHi was 7.25 ± 0.04 and 7.15 ± 0.05 in control and hydrocortisone‐treated cells, respectively (0.1 < P < 0.05). Following intracellular acidification in the absence of external Na+, pHi recovery upon addition of Na+ was increased 89% in hydrocortisone‐treated cells relative to control. This was due to an increase in the Vmax for the Na+/H+ exchanger from 17.5 ± 2.4 to 25.9 ± 2.0 nmol Na+/mg protein × min (P < 0.01) without a significant change in Km. Treatment of VSMC with actinomycin D (1 μg/ml) or cycloheximide (10 μM) completely inhibited the hydrocortisone‐mediated increase in Na+/H+ exchange, indicating a requirement for both RNA and protein synthesis. Because hydrocortisone altered the Vmax for Na+/H+ exchange, in contrast to agonists such as serum or angiotensin II which alter the Km for intracellular H+ or extracellular Na+, respectively, we studied the effect of hydrocortisone on activation of Na+/H+ exchange by these agonists. In cells maintained at physiological pHi (7.2), the initial rate (2 min) of angiotensin II‐stimulated alkalinization was increased 66 ± 39% in hydrocortisone‐treated compared with control cells. Hydrocortisone caused no change in angiotensin II‐stimulated phospholipase C activity assayed by measurement of changes in intracellular Ca2+ or diacylglycerol formation. However, angiotensin II and serum stimulated only small increases in Na+/H+ exchange in acid‐loaded (pHi = 6.8) hydrocortisone‐treated cells. These findings suggest that hydrocortisone‐mediated increases in VSMC Na+/H+ exchange occur in association with a nonproliferating phenotype that has altered regulation of Na+/H+ exchange activation. We propose that hydrocortisone‐mediated growth inhibition may be a useful model for studying the role of Na+/H+ exchange in cell growth responsiveness.
Lack of proteolytic processing of α‐L‐fucosidase in human skin fibroblasts
Authors: Leibold Debra M. , Robinson Cynthia B. , Scanlin Thomas F. , Glick Mary Catherine
Published: February 2005

Acid hydrolases are synthesized as precursors that undergo several posttranslational modifications including proteolytic processing to a smaller mature enzyme. The amount of proteolytic processing varies for different acid hydrolases, and many details of the intracellular pathways are not known. The processing of α‐L‐fucosidase was distinguished from that of other acid hydrolases reported when studied in systematic pulse‐chase labeling experiments. Only one form of α‐L‐fucosidase, Mr 56,000‐57,000, was demonstrated intra‐ and extracellularly. Under the same conditions, N‐acetyl‐β‐D‐glucosaminidase was shown to be processed with several forms, as previously reported by Hasilik and Neufeld (1980a). To obtain these results, human skin fibroblasts were labeled metabolically with L‐[3H]leucine for periods of 20 min to 8 hr with varying periods of chase from 1 to 96 hr with nonradioactive L‐leucine. α‐L‐Fucosidase was immunoprecipitated by a polyclonal antibody from material extracted from cells and ammonium sulfate precipitated medium and was examined by polyacrylamide gel electrophoresis under denaturing conditions. N‐Acetyl‐β‐D‐glucosaminidase was examined with similar procedures and served as a control for the methods. Tunicamycin treatment of the cells was used to show that glycosylation did not obscure proteolytic processing because, again, only one form of the intra‐ and extracellular enzyme was observed, although of smaller size, Mr 52,000‐53,000. In addition, separation of the cells into prelysosomal and lysosomal fractions showed only one form of the enzyme. It is concluded that α‐L‐fucosidase does not undergo proteolytic processing in human skin fibroblasts in the usual manner described for other acid hydrolases.
Enhancement of phorbol ester‐induced protein kinase activity in human neutrophils by platelet‐activating factor
Authors: Gay James C. , Stitt Ella S.
Published: February 2005

We have shown that platelet‐activating factor (PAF), a weak primary stimulus for neutrophil superoxide generation, synergistically enhances neutrophil oxidative responses to the tumor promoter phorbol myristate acetate (PMA). Since PMA is known to cause cytosol‐to‐membrane shift of calcium‐activated, phospholipid‐dependent protein kinase (protein kinase c, PKC) in human neutrophils, we investigated the role of PAF in modifying PMA‐induced PKC activation/translocation. Protein kinase activity was measured as the incorporation of 32P from γ‐32P‐ATP into histone H1 induced by enzyme in cytosolic and particulate fractions from sonicated human neutrophils. PAF did not alter the sharp decrease in cytosolic PKC activity induced by PMA. However, in the presence of PAF and PMA, total particulate protein kinase activity increased markedly over that detected in the presence of PMA alone (144 ± 9 pmoles 32P/107PMN/minute in cells treated with 20 ng/ml PMA compared to 267 ± 24 pmoles 32P in cells exposed to PMA and 10−6M PAF). The increase in total particulate protein kinase activity was synergistic for the two stimuli, required the presence of cytochalasin B during stimulation, and occurred at PAF concentration of 10−7and M above. Both PKC and calcium‐, phospholipid‐independent protein kinase activities in whole particulate fractions were augmented by PAF as were both activities in detergent‐extractable particulate subfractions. PAF did not directly activate PKC obtained from control or PMA‐treated neutrophils. However, the PKC‐enhancing effect of PAF was inhibited in the absence of calcium during cellular stimulation. PAF also increased particulate protein kinase activity in cells simultaneously exposed to FMLP but the effect was additive for these stimuli. These results suggest that PAF enhances PMA‐induced particulate PKC activity by a calcium‐dependent mechanism. The enhancing effect of PAF may be directly involved in the mechanism whereby the phospholipid “primes” neutrophils for augmented oxidative responses to PMA.
Resumption of cell cycle in Balb/c‐3T3 fibroblasts arrested by polyamine depletion: Relation with “Competence” gene expression
Authors: Charollais R. H. , Mester J.
Published: February 2005

Serum deprivation arrests Balb/c‐3T3 fibroblasts (clone A31) in G0 phase, where resumption of the cell division cycle can be induced by addition of serum or of specific growth factors in a defined sequence: PDGF (inducer of a state of “competence,” characterized by the expression of a family of genes including c‐myc), epidermal growth factor EGF and IGF1 (Leof et al., 1982, 1983). When exponentially growing A31 cells are placed for ≥ 2 days in a medium containing the alpha‐difluoromethylornithine (αDFMO), an irreversible inhibitor of ornithine decarboxylase, they become arrested in G1 phase as a consequence of polyamine depletion (Medrano et al., 1983). In the αDFMO‐arrested cells, addition of putrescine (60 μM) in a culture medium containing 6% fetal calf serum (FCS), but not in serum‐free medium, is sufficient to induce G1 progression and entry into S phase (as determined by 3H‐thymidine incorporation). The level of “competence” mRNAs is high in αDFMO‐arrested cells. After addition of putrescine in FCS‐containing medium, these mRNAs continue to be present for at least 3 h. A large proportion of αDFMO‐arrested cells can be induced to progress to S phase by insulin (1 μM, acting via IGF1 receptor) plus putrescine in a serum‐free medium (≥ 50% of FCS effect). In this case, the levels of “competence” mRNAs become low or undetectable within 3 h, EGF (10 nM) plus insulin had only slightly greater effect than insulin alone on the progression of αDFMO‐arrested cells. When the αDFMO‐arrested cells are subsequently incubated during 3 days in a low‐serum‐containing medium (0.25% FCS), they do not replenish their supply of polyamines, and then continue to express the c‐myc gene. The recruitment of the polyamine‐depleted, serum‐deprived cells into the cell division cycle does not require PDGF and can be induced by addition of EGF and insulin plus putrescine. These data indicate that αDFMO arrests majority of the cells at a point situated beyond the PDGF‐ and EGF‐dependent portion of G1 phase. During the subsequent serum deprivation, the αDFMO‐arrested cells remain “competent” (PDGF‐independent), probably as a consequence of their continued expression of c‐myc (and possibly other PDGF‐inducible genes).
Stimulation of cell growth and inhibition of prostacyclin production by heparin in human umbilical vein endothelial cells
Authors: Hasegawa N. , Yamamoto M. , Yamamoto K.
Published: February 2005

We characterized human umbilical vein (HUV) endothelial cells as to cell growth and prostacyclin production to get a better understanding of the properties of endothelial cells. Endothelial cell growth supplement (ECGS) and basic fibroblast growth factor (FGF) stimulated HUV endothelial cell growth. Heparin further enhanced the cell growth stimulated by ECGS, but not the cell growth stimulated by FGF or in the absence of these growth factors. In the presence of ECGS, the prostacyclin‐producing capacity of the cells was inhibited by heparin. However, in the presence of FGF of in the absence of growth factors, heparin did not inhibit prostacyclin production. Therefore, it is likely that there is a specific correlation between heparin and growth factors for endothelial cells in the blood vessel to maintain nonthrombogenicity properly. Heparin‐treated cultures may not be suitable for some examinations of prostacyclin production by vascular endothelial cells.
Differential stimulation of collagenase and chemotactic activity in fibroblasts derived from rat wound repair tissue and human skin by growth factors
Authors: Buckley‐Sturrock Anne , Woodward Stephen C. , Senior Robert M. , Griffin Gail L. , Klagsbrun Michael , Davidson Jeffrey M.
Published: February 2005

Epidermal growth factor and cartilage‐derived basic fibroblast growth factor (EGF and CD‐bFGF) are mitogens shown to increase the rate of wound repair in animal models. In addition to being a mitogen for granulation tissue, CD‐bFGF stimulates the recruitment of cells to the wound site. CD‐bFGF and a closely‐related chondrosarcoma‐derived fibroblast growth factor stimulated chemotaxis of granulation tissue cells in vitro, each factor having a maximum activity at a concentration of 55 pM. Epidermal growth factor was also a potent chemoattractant for rat granulation tissue fibroblasts; however, maximum activity was obtained at 1.7 nM. Cells from all stages of wound repair were chemotactically responsive to these factors, but there was some attenuation of the response to bFGF in cells derived from fully‐organized day 28 granulation tissue. Collagenase‐catalyzed restructuring of collagen, an additional significant feature of wound repair, is probably critical to cell movement in an extracellular matrix. Cells derived from organizing (6–day old) sponge granulation tissue secreted latent collagenase constituitively in vitro. In the presence of serum, the production of collagenase was stimulated three‐four fold by 1.8 nM bFGF derived either from cartilage or chondrosarcoma. When serum was present, as at a wound site, collagenase production was not enhanced by the addition of EGF. Cells from fully organized, day 21 sponge granulation tissue did not secrete latent collagenase constitutively and could not be stimulated to do so by the addition of EGF, bFGF, or phorbol ester. Human skin fibroblast collagenase production was also stimulated by bFGF and was refractory to EGF. While both classes of growth factor have the ability to promote wound healing, the varying responses they elicit in cell populations from the wound site emphasize the different pathways of cellular activation.
Vasoconstrictor hormones depolarize renal glomerular mesangial cells by activating chloride channels
Authors: Kremer Sid G. , Breuer William V. , Skorecki Karl L.
Published: February 2005

Mesangial cells are smooth muscle‐like cells of the renal glomerulus which contract and produce prostaglandins in response to vasopressin and angiotensin. These responses serve to regulate the glomerular capillary filtering surface area. We have used the membrane potential‐sensitive fluorescent dye bis‐oxonol and the intracellular fluorescent calcium‐sensitive probe Indo‐1 to study the changes in membrane potential (Em) and intracellular free calcium concentration ([Ca2+ ]i) in cultured rat mesangial cells in response to vasoconstrictor hormones. Basal [Ca2+]i was 227 ± 4 nM, and stimulation by maximal concentrations of either vasopressin or angiotensin resulted in a transient 4–6‐fold rise. Resting membrane potential was 45.8 ± 0.9 mV and vasoconstrictor hormones caused a depolarization of 14–18 mV. The following extracellular ion substitutions indicated that chloride efflux was the predominant ion flux responsible for depolarization: (1) depolarization persisted when sodium in the medium was substituted with N‐methylglucamine; (2) substitution of medium sodium chloride with sodium gluconate, which enhances the gradient for chloride efflux, augmented vasoconstrictor‐stimulated depolarization; (3) suspension of cells in potassium chloride medium resulted in depolarization, following which, stimulation by either vasopressin or angiotensin resulted in hyperpolarization; and (4) this hyperpolarization did not occur when potassium gluconate medium was used to depolarize the cells. The calcium ionophore ionomycin also resulted in membrane depolarization. However, prevention of the rise in [Ca2+]i by prior exposure to ionomycin in calcium‐free medium or by loading mesangial cells with the intracellular calcium buffer BAPTA did not abrogate the depolarization response to vasoconstrictor hormones. This indicates that a rise in intracellular calcium is not necessary for depolarization. In contrast, prior depolarization of the cells using varying concentrations of KCl in the external medium, which dissipated the electrochemical gradient for chloride efflux, resulted in a corresponding prolongation of the transient calcium response to vasopressin and angiotensin. These findings indicate that angiotensin and vasopressin depolarize mesangial cells by activating chloride channels and that this activation can occur by both calcium‐dependent and ‐independent mechanisms. In addition, activation of chloride channels with resulting depolarization may serve to modulate the calcium signal.
Serotonin uptake and configurational change of bovine pulmonary artery smooth muscle cells in culture
Authors: Lee S.‐L. , Dunn J. , Yu F.‐S. , Fanburg B. L.
Published: February 2005

Although it is well known that endothelial cells transport serotonin (5‐HT) from extracellular to intracellular locations, it has been generally assumed that smooth muscle cells do not accumulate 5‐HT but, rather, respond to 5‐HT through a receptor activity unrelated to uptake of this amine or via stimulation of endothelial‐derived relaxing factor. In the present study smooth muscle cells (PASMC), isolated and cultured from bovine pulmonary artery, were evaluated for 5‐HT uptake under a variety of conditions. 5‐HT uptake was linear up to 15 min and the rate was seven‐ to eightfold higher than that by bovine pulmonary artery endothelial cells. There was intracellular metabolism of 5‐HT to 5‐hydroxy‐indoleacetic acid (5‐HIAA). The uptake was inhibited by exposure to 4°C, absence of Na+ from the medium, and agents such as imipramine, verapamil, ketanserin, and methiothepin. Like that of endothelial cells, 5‐HT uptake by PASMC was stimulated by exposure of cells to anoxia for 24 hr. Unlike endothelial cells that showed no morphological changes, PASMC at early passage showed dendritic formation after 30–60 min exposure to 5‐HT at a concentration as low as 10−8 M. Although this configurational change in response to 5‐HT was lost with passage of cells, transport of 5‐HT by these cells was retained. The configurational change was blocked by agents that inhibited 5‐HT uptake, such as imipramine, verapamil, ketanserin, and methiothepin; it was unaffected by inhibitors of protein kinase C, phospholipase C, and calmodulin or absence of Ca2+ from the medium. We conclude that PASMC, as well as endothelial cells, accumulate 5‐HT; there appears to be a close relationship between 5‐HT uptake and configurational change of early passaged PASMC in culture. The factor(s) required for the configurational change are absent in endothelial cells and lost during passage of PASMC.
Defective control of cytoplasmic calcium concentration in T lymphocytes from old mice
Authors: Miller Richard A. , Philosophe Ben , Ginis Irene , Weil Gary , Jacobson Bruce
Published: February 2005

Cytoplasmic calcium concentration ([Ca]i) rises within minutes of exposure of T lymphocytes to a mitogen. T cells from old mice are defective in this reaction, a defect that could reflect either altered signal transduction or instead a more general age‐associated change in intracellular calcium regulation. We therefore tested the ability of T cells from old mice to regulate their [Ca]i concentration after exposure to low concentrations of ionomycin, an agent that raises [Ca]i but bypasses receptor‐mediated signal transduction mechanisms. Exposure of T cells to ionomycin leads to an abrupt increase in [Ca]i followed by stabilization at a dose‐dependent plateau level that is affected by extracellular EGTA, by calmodulin inhibitors, and by modulators of protein kinase C. Plateau levels of [Ca]i after ionomycin challenge were consistently lower in T cells from old mice than in T cells from young mice. Flow cytometric experiments showed that while essentially all T cells from both old and young mice responded to ionomycin, they did so to an extent that depended on donor age. The age‐dependent increase in resistance to ionomycin‐induced changes in [Ca]i cannot be attributed to diminished membrane permeability to the ionomycin‐calcium complex. The data suggest that aging may lead, in T lymphocytes, to a relative resistance to increases in [Ca]i, a resistance that in turn prevents cell activation.
Coordination of keratinocyte programming in human SCC‐13 squamous carcinoma and normal epidermal cells
Authors: Rubin Andrew L. , Parenteau Nancy L. , Rice Robert H.
Published: February 2005

Exploiting the sensitivity of neoplastic keratinocytes to physiological effectors, this work analyzes the degree of coordination among differentiation markers in the established human epidermal squamous carcinoma cell line SCC‐13 in comparison to normal human epidermal cells. This analysis showed that overall keratin content was modulated substantially and in parallel with particulate transglutaminase activity in response to variation of calcium, retinoic acid, and hydrocortisone concentrations in the medium. The changes in keratin expression were evident primarily in the striking stimulation by hydrocortisone or calcium and the virtual suppression by retinoic acid of species in the 56–58 kd region, which have not previously been reported subject to such physiological modulation. In contrast, involucrin levels were coordinated only to a limited degree with particulate transglutaminase activity and keratin content. The very low involucrin levels observed in low calcium medium were increased 5‐ to 10‐fold in high calcium medium. However, they were also increased 5‐ to 30‐fold in low calcium medium by retinoic acid, a clear example of uncoupling. Activities of the tissue transglutaminase were altered considerably by the various culture conditions but were not obviously coordinated to keratinocyte markers. In normal epidermal cells, the suppressive effect of retinoic acid was much more evident with particulate transglutaminase than involucrin levels. While calcium had a large stimulatory effect on both markers, hydrocortisone had little or no influence. These results emphasize the potential importance of quantitative analysis of differentiation markers for resolving the contribution of physiological elements in coordination of cellular programming.
Common pathway for the induction of hexose transport by insulin and stress
Authors: Warren A. P. , Pasternak C. A.
Published: February 2005

The effect of stress (heat shock, arsenite, or Semliki Forest virus [SFV] infection) on the induction of increased hexose transport has been compared with that of insulin. All four treatments increase the Vmax for transport by BHK cells three‐to five‐fold, with little effect ( < 40% decrease) on Km. Hydrogen peroxide and phenylarsine oxide (PAO) prevent the increase in hexose transport induced by stress treatments as effectively as they do that induced by insulin. Pinocytosis is not affected by any of the four treatments. On the other hand, the induction by insulin is sensitive to amiloride, whereas that by arsenite is not. Rat embryo fibroblasts, which respond poorly to insulin, respond well to arsenite, heat shock, or SFV infection. It is concluded that the stress response is mediated by certain compounds that may be common to those required for the action of insulin, but that those compounds act at a stage subsequent to the function of the insulin receptor.
Regulation of hexose transport in rat myoblasts during growth and differentiation
Authors: Chen S. R. , Lo T. C. Y.
Published: February 2005

We report here the effects of growth conditions and myogenic differentiation on rat myoblast hexose transport activities. We have previously shown that in undifferentiated myoblasts the preferred substrates for the high (HAHT)‐ and low (LAHT)‐affinity hexose transport systems are 2‐deoxyglucose (2‐DG) and 3‐O‐methyl‐D‐glucose (3‐OMG), respectively. The present study shows that at cell density higher than 4.4 × 104 cells/cm2, the activities of both transport processes decrease with increasing cell densities of the undifferentiated myoblasts. Since the transport affinities are not altered, the observed decrease is compatible with the notion that the number of functional hexose transporters may be decreased in the plasma membrane. Myogenic differentiation is found to alter the 2‐DG, but not the 3‐OMG, transport affinity. The Km values of 2‐DG uptake are elevated upon the onset of fusion and are directly proportional to the extent of fusion. This relationship between myogenesis and hexose transport is further explored by using cultures impaired in myogenesis. Treatment of cells with 5‐bromo‐2′‐deoxyuridine abolishes not only myogenesis but also the myogenesis‐induced change in 2‐DG transport affinity. Similarly, alteration in 2‐DG transport affinity cannot be observed in a myogenesis‐defective mutant, D1. However, under myogenesis‐permissive condition, the myogenesis of this mutant is also accompanied by changes in its 2‐DG transport affinity. The myotube 2‐DG transport system also differs from its myoblast counterpart in its response to sulfhydryl reagents and in its turnover rate. It may be surmised from the above observations that myogenesis results in the alteration of the turnover rate or in the modification of the 2‐DG transport system. Although glucose starvation has no effect on myogenesis, it is found to alter the substrate specificity and transport capacity of HAHT. In conclusion, the present study shows that hexose transport in rat myoblasts is very sensitive to the growth conditions and the stages of differentiation of the cultures. This may explain why different hexose transport properties have been observed with myoblasts grown under different conditions.
Regrowth kinetics of cells from different regions of multicellular spheroids of four cell lines
Authors: Freyer James P. , Schor Patricia L.
Published: February 2005

A basic understanding of the recruitment of quiescent tumor cells into the cell would be an important contribution to tumor biology and therapy. As a first step in pursuing this goal, we have investigated the regrowth kinetics of cells from different regions in multicellular spheroids of rodent and human origin. Cells were isolated from four different depths within the spheroids using a selective dissociation techinque. The outer cells were proliferating and resumed growth after replating with a 0–8‐hour lag period, similar to cells from exponentially growing monolayers. with increasing depth of origin, the lag periods prior to regrowth increased to 2–3 times the monolayer doubling time; cells from plateau‐phase monolayers showed a lag period of 1–1.5 times the doubling period. After resuming grwoth, all cells of a given cell line grew with the same doubling time and achieved the same confluency level. The inner spheroid cells and cells from plateau‐phase monolayers had reduced clonogenic efficiencies. The inner cells were initially 1.5–3 times smaller than the outer cells, but began to increase in volume within 4 hours of replating. The fractions of S‐phase cells were greatly decreased with increasing depth of origin in the spheroids; there were long delays prior to s‐phase recovery after plating, to a maximum of 1–1.5 times the normal doubling time. These results suggest that those quiescent cells from spheroids and monolayers which are able ot reenter the cell cycle are predominantly in the G1‐phase. However, quiescent cells from the innemost spheroid region require approximately twice as long ot enter normal cell cycle traverse as cells from plateau‐phase monolayers. The selective dissociation method can isolate very pure populations of proliferating and quiescent cells in a rapid and nonpeturbing manner; this system will be valuable in further characterizing quiescent cells from spheroids.
A mammary‐derived growth inhibitor (MDGI) related 70 kDa antigen identified in nuclei of mammary epithelial cells
Authors: Müller T. , Kurtz A. , Vogel F. , Breter H. , Schneider F. , Angström U. , Mieth M. , Böhmer F.‐D. , GrosseVogel R.
Published: February 2005

The aim of the present study was to investigate the expression of the mammary derived growth inhibitor (MDGI) and the subcellular localization of MDGI related antigens in bovine mammary glands. Cell‐free translation of poly (A+) = RNA, immunoprecipitation with rabbit anti‐MDGI‐antibodies, and estimation of the relative contents of MDGI by a radioimmunoassay in mammary tissue of different functional statess revealed that the 13 kDa MDGI was dramatically increased in terminally differentiated mammary tissue compared with the proliferating tissue from pregnant animals. To address the question of tissue localizationl, polyclonal anti‐MDGI antibodies and antibodies directed aganist a sythetic peptide corresponding ot residues 69 to 78 of MDGI were used. Western blotting of tissue fractions revealed the cytosolic and microsomal localization of MDGI. Additionally, both types of antibodies a 70‐kDa antigeninthe unclear fractionof differentiated mammary glands. Salt extraction and DNase I digestion of isolated unclei, as well as chromatin purification, indicated an association of the 70‐kDa antigen with the chromatin. By means of the immunogold technique, MDGI‐related antigens were localized within euchromatic unclear regions of epithelial cells in the intact differentiated mammary gland. The immunostaining was markedly diminished in the proliferating tissue. This finding raises the possibility that MDGI and the 70‐kDa antigen influence cell proliferation by acting on geneexpression within the unclei of mammary glands.
Heparin interactions with cultured human vascular endothelial and smooth muscle cells: Incidence on vascular smooth muscle cell proliferation
Authors: Herbert Jean‐Marc , Maffrand Jean‐Pierre
Published: February 2005

The binding, internalization, and metabolism of [3H]‐heparin by human umbilical vein endothelial cells (HUVEC) and human umbilical arterial smooth muscle cells (HUASMC) have been characterized using size‐exclusion HPLC. Incubation of HUVEC awith [3H]‐heparin demonstrated selective binding of high‐molecular‐weight (MW) components (MW = 21 kd), which was followed by rapid, temperature‐dependent internalization. Over the next 3 hours, this internalized [3H]‐heparin was degraded to low‐MW fragments (MW = 0.9 kd). Primary cultures of HUASMC selectively bound extremely high‐MW components (MW = 40 kd) and also smaller components whose MW (0.9 kd) corresponded to that of the heparin metabolite(s) formed by HUVEC. Subcultured HUASMC bound only the 40‐kd components. Internalization of heparin by smooth muscle cells (SMC) was significantly slower than that determined for HUVEC, and even after 4 hours there was no evidence of the heparin being metabolized. However, when incubating primary rabbit aortic SMC with purified low‐MW heparin fragment(s) produced by culture by HUVEC, a significantly lower proliferative response of these cells (IC50 = 18.4 μg/ml) was obtained. Virtually no effect was observed with subcultured SMC in the range of the tested concentrations (0–20 μg/ml). These fragments were 10‐ to 15‐fold more effective in inhibiting primary SMC growth than was standard heparin. Furthermore, heparin fractions in the same range of molecular weights, purified either after nitrous acid or heparinase depolymerization of standard heparin, showed no activity on primary SMC growth, thus indicating a high degree of selectivity of the heparin metabolite(s) produced by HUVEC in culture.
Metabolism of small RNAs in cultured human cells
Authors: Choudhury Kanakendu , Choudhury Indrani , Eliceiri George L.
Published: February 2005

There are gaps in what is known about the metabolism of some mammalian small RNA species. Our present observations can be summarized as follows. The level of radiolabeled mature U1 RNA doubled between 2 and 24 hr of label chase, while that of all other small RNA species tested did not change. These results are compatible with the possibility that the nucleotide precursor pool for U1 RNA transcription may be partly segregated, or that there may be a second pathway of U1 RNA formation. Precursors of nucleolar U3 RNA were detected whose electrophoretic mobilities are equivalent to those of transcripts ∼ 14 and ∼ 8 nucleotides longer than the mature species, and which are apparently cytoplasmic. The ladder of U2 RNA precursors showed a gap, suggesting that some of the cleavages during U2 RNA processing are endonucleolytic. We detected an apparent U5 RNA precursor whose electrophoretic mobility is equivalent to that of a species ∼ 1 nucleotide longer than mature U5 RNA. There was a predominant band in the middle of the ladder of U4 RNA precursors (apparently ∼ 3 nucleotides longer than mature U4 RNA) which suggests that U4 RNA maturation may pause briefly at that intermediate. There are apparently two additional species of mature hY3 RNA, which are less abundant and are about 1 and 2 bases longer than the major hY3 RNA species. An apparent hY3 RNA precursor was detected, which may be ∼ 2–3 nucleotides longer than the main mature hY3 RNA species.
Effects of vanadate on tyrosine phosphorylation and the pattern of glycosaminoglycan synthesis in rabbit chondrocytes in culture
Authors: Owada M. Koji , Iwamoto Masahiro , Koike Tatsuya , Kato Yukio
Published: February 2005

The present study examined the effects of high doses of vanadate on glycosami‐noglycan (GAG) synthesis and tyrosine phosphorylation in rabbit chondrocytes in confluent cultures. Although 6 μM vanadate increased the incorporation of [3H] glucosamine into chondroitin sulfate proteoglycans twofold, 40–60 μM vanadate suppressed this incorporation fourfold. Although 6 μM vanadate had little effect on [3H] glucosamine incorporation into hyaluronate, 40–60 μM vanadate increased this incorporation threefold. Chemical analyses confirmed that the increase in ∥3H∥glucosamine incorporation into hyaluronate and the decrease in the incorporation into chondroitin sulfate proteoglycan correlated with increased hyaluronate content and decreased chondroitin sulfate content in the cell layers of vanadate‐transformed cells. Chondrocytes exposed to 40–60 μM vanadaje became typically transformed spindlelike cells. Furthermore, vanadate, at 6 and 60 μM, increased the overall level of phosphotyrosine by 8‐ and 31‐fold, respectively, and 60 μM vanadate enhanced phosphorylation of many phosphotyrosine‐containing proteins. These observations suggest that vanadate induces transformation‐associated changes in the pattern of GAG synthesis when it induces excess phosphorylation on tyrosine in chondrocyte proteins.
Dynamic interactions of c‐fos protein in serum‐stimulated 3T3 cells
Authors: Vosatka Robert J. , Hermanowski‐Vosatk Anne , Metz Richard , Ziff Edward B.
Published: February 2005

The c‐fos gene, the cellular homologue of the transforming gene of the FBJ osteosarcoma virus, v‐fos, is strongly induced in quiescent BALB/c 3T3 cells by growth factors and in other cell types by a wide variety of transmembrane signalling agents. c‐fos is a member of a family of structurally related proteins which includes the fos‐related antigens (fra). We have studied the dynamic state of the c‐fos protein with an antibody prepared by immunizing rabbits with a plasmid‐encoded fos fusion protein. In serum‐stimulated BALB/c 3T3 cells, the antibody recognizes a nuclear antigen which resolves on SDS‐PAGE as a 60–68‐kD group of bands corresponding to c‐fos, a doublet at 44–45‐kD corresponding to the noncovalently associated p39 protein, as well as an approximately 50‐kD band corresponding to a fra. We show that although c‐fos protein synthesis is only transiently induced by serum, the c‐fos protein persists within the cell after its synthesis has ceased, and it decays with a half‐life of 2 hours. Significantly, newly synthesized p39 continues to appear in the immune‐precipitated complex even at times when c‐fos is no longer synthesized. These kinetics indicate that even following shutoff of c‐fos protein synthesis, p39 is newly synthesized and can complex with c‐fos protein or a fos‐related antigen. During this time, c‐fos also undergoes an extensive posttranslational modification. The modification is partially reversed by phosphatase treatment, which implicates protein phosphorylation. Together these results suggest that both interaction with p39 and phosphorylation may progressively modify the properties of c‐fos and/or the fos‐related antigens over a period of 4–8 hours following the shutoff of fos synthesis. We discuss the implications of the dynamic state of c‐fos and fra protein interactions for the function of these proteins.