bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

AbstractWe 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

AbstractAcid 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

AbstractWe 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

AbstractSerum 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

AbstractWe 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

AbstractEpidermal 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

AbstractMesangial 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

AbstractAlthough 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

AbstractCytoplasmic 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

AbstractExploiting 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

AbstractThe 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.