bluuebunny/crossref_metadata_2025 huggingface dataset:
β‐adrenergic control of cell volume and chloride transport in an established rat submandibular cell line
Authors: He Xinjun , Ship Jonathan , Wu Xiaozai , Brown Ashley M. , Wellner Robert B.
Published: February 2005

Rat submandibular cells treated with methylcholanthrene are able to be propagated in continuous culture while retaining β‐adrenergic responsiveness. A specific clone, RSMT‐A5, has been isolated and studied in detail. RSMT‐A5 cells possess β‐adrnergic receptors (BARS) as judged by [3H]‐dihydroalprenolol ([3H]‐DHA) binding studies. [3H]‐DHA binds to RSMT‐A5 membranes in a specific and saturable manner with respect to time and [3H]‐DHA concentration. Specific binding is saturable within three min of incubation, and a Scatchard analysis reveals a single class of high affinity binding sites with an equilibrium dissociation constant of 0.62 ± 0.03 nM and a receptor density of 101 ± 4 fmole/mg protein. Antagonist competition studies indicate that the BARs are primarily of the β2‐subtype. The BARs are functional since isoproterenol stimulation results in an increased intracellular cAMP content, marked morphological change, and decreased cell volume and chloride content. These same responses can be evoked by treating RSMT‐A5 cells with 8‐bromo‐cAMP. Ion transport inhibitors such as bumetanide (an inhibitor of Na/K/C1 cotransport), SITS and DIDS (inhibitors of chloride‐bicarbonate exchange), amiloride (an inhibitor of Na‐H exchange), ouabain (an inhibitor of Na/K‐ATPase), and dipyridamole and 9‐anthracene carboxylic acid (chloride channel blockers) fail to inhibit the isoproterenol‐stimulated change in chloride content. The effects of either isopro‐terenol or 8‐bromo‐cAMP on both chloride content and cell volume can be inhibited by the chloride channel blocker N‐phenylanthranilic acid, however. Taken together, our results indicate that RSMT‐A5 cells possess a β‐adrenergic receptor system which controls intracellular volume and chloride content by modulating transport processes that are 1) cAMP‐responsive and 2) inhibitable by the putative chloride channel blocker N‐phenylanthranilic acid.
Protein synthesis during induction of DNA replication in thyroid epithelial cells: Evidence for late markers of distinct mitogenic pathways
Authors: Lamy Françoise , Roger Pierre , Lecocq Raymond , Dumont Jacques E.
Published: February 2005

The synthesis of specific proteins has been investigated in primary cultures of dog thyroid epithelial cells, which can be induced to progress into G1 phase, in the presence of insulin, by different types of mitogens: thyrotropin (TSH) acting through cyclic adenosine monophosphate (cAMP), epidermal growth factor (EGF), 12‐O‐tetradecanoyl‐phorbol‐13‐acetate (TPA), or 10% serum. EGF, TPA, or serum specifically induce [35S] methionine labeling of protein 1 (Mr ≈ 80,000). The effect of EGF on protein 1 labeling and DNA replication is dependent on insulin. The level of protein 1 labeling as well as that of DNA synthesis is higher when TSH or TSH + serum are added together with EGF. It peaks in mid‐G1. TSH alone, in the presence of insulin, stimulates DNA replication without inducing protein 1 synthesis, which thus represents a cell‐cycle‐dependent event that is not obligatory in mitogenic activation through cyclic AMP. Among the eight proteins whose synthesis is stimulated by TSH, only the labeling of protein 7, molecular weight ratio (Mr ≈ 38,000), correlates with the DNA synthetic activity of the cells. The present authors identified protein 7 as cyclin/proliferating cell nuclear antigen (PCNA), the auxiliary protein of DNA polymerase‐δ. The effect of TSH on cyclin synthesis is already detectable when most of the cells are in late G1, but its stimulation by EGF or EGF + serum is delayed and detected only after extending the labeling period to the S‐phase. These data support the view that the cAMP‐mediated mitogenic pathway remains partly distinct from the better known pathways induced by growth factors and tumor promoters, even at late stages of the G1‐phase.
An epidermal growth factor‐ricin a chain (EGF‐RTA)‐resistant mutant and an epidermal growth factor‐Pseudomonas endotoxin (EGF‐PE)‐resistant mutant have distinct phenotypes
Authors: Banker Deborah E. , Pastan Ira , Gottesman Michael M. , Herschman Harvey R.
Published: February 2005

H20e12 is a mutant HeLa cell line selected for resistance to the toxicity of a chimeric protein conjugate composed of epidermal growth factor (EGF) and the toxic A cham of ricin (RTA). ET‐28 is a mutant KB cell line selected for resistance to the toxicity of a chimeric protein conjugate composed of EGF and Pseudomonas exotoxin (PE). In this report we describe the presence or absence, in these mutants, of cross‐resistance to the two toxic conjugates and the effects of ammonium chloride, leupeptin, and adenovirus cotreatments on toxin efficacies. ET‐28 cells, the EGF‐PE‐resistant cells, are resistant to both EGF‐PE and EGF‐RTA. In contrast, H20e12 cells, the EGF‐RTA‐resistant cells, are as sensitive to EGF‐PE toxicity as are their parent HeLa cells. Ammonium chloride cotreatment substantially reduces the resistance of H20e12 cells to EGF‐RTA but has little or no effect on the resistance of ET‐28 cells to either EGF‐RTA or EGF‐PE. Leupeptin has no effect on the toxicity of either chimeric conjugate on any of the four cell lines, despite its demonstrated ability to inhibit cellular degradation of EGF. In contrast, adenovirus cotreatment enhances the toxicity of EGF‐RTA and EGF‐PE on all cells tested, and completely nullifies the relative resistance of H20e12 and ET‐28 cells to these toxic conjugates. H20e12 and ET‐28 cells appear to be altered in distinct, possibly endosomal, functions.
Multiple intracellular pathways induce expression of a zinc‐finger encoding gene (EGR1): Relationship to activation of the Na/H exchanger
Authors: Jamieson Gordon A. , Mayforth Ruth D. , Villereal Mitchel L. , Sukhatme Vikas P.
Published: February 2005

Intracellular pathways that rapidly stimulate the expression of a mitogeninducible, zinc‐finger encoding gene, EGR1 (Sukhatme et al., Cell 53:37–43), have been characterized in two human fibroblasts strains (WI‐38 and HSWP). Serum and epidermal growth factor (EGF) were each found to strongly stimulate EGR1 expression in both cell types. Comparably high levels of expression could also be induced by treatment with the phorbol ester TPA. In cells rendered deficient in PK‐C, serum and EGF were each still capable of inducing high levels of EGR1 mRNA. demonstrating that additional non‐protein kinase C pathways are capable of stimulating EGR1 expression. In both fibroblasts strains, stimulation of EGR1 expression by all these agents exhibited rapid, transient kinetics and could be superinduced if protein synthesis was inhibited through the addition of cycloheximide. Finally, various agents, known to stimulate/inhibit the activation of another early mitogenic response, the activation of Na/H exchange, were analyzed for their effect on EGR1 expression. Interestingly bradykinin, vasopressin, and Ca ionophores, which dramatically stimulate Na/H exchange, were only weak stimulants of EGR1 expression. Conversely, EGF, which stimulates Na/H exchange poorly, strongly activated EGR1 expression. Hence while EGR1 expression could be triggered by multiple intracellular pathways, its expression does not appear to require the prior activation of Na/H exchange.
Proliferation of fetal rat hepatocytes in response to growth factors and hormones in primary culture
Authors: Hoffmann Birgit , Piasecki Angelika , Paul Dieter
Published: February 2005

Fetal rat hepatocytes (day 19 of gestation) multiply in primary culture in arginine‐free, hydrocortisone‐containing chemically defined medium MX‐82 supplemented either with epidermal growth factor (EGF) or insulin or both. In contrast, hepatocytes did not multiply under similar culture conditions using Dulbecco's minimum essential medium (DMEM). Cells underwent two divisions within 10 days in cultures maintained in MX‐82 medium without a medium change, and cells grew to increased final cell densities when the medium was renewed every third day. When the medium MX‐82 was enriched by the addition of lipids, intermediary metabolites, and trace metals (medium MX‐83), cells grew to higher densities. In the absence of the growth factors, cells became quiescent and subsequently could be induced to synthesize DNA in response to EGF. With the increasing numbers of cells per dish, the growth response of the hepatocytes diminished. Levels of hepatocyte‐specific albumin and α‐fetoprotein mRNAs at day 0 were similar to those observed at day 10 in primary fetal rat hepatocyte cultures and were maintained at higher levels in medium MX‐83 than in medium MX‐82.
Dexamethasone effects on creatine kinase activity and insulin‐like growth factor receptors in cultured muscle cells
Authors: Whitson Peggy A. , Stuart Charles A. , Huls M. Helen , Sams Clarence F. , Cintron Nitza M.
Published: February 2005

We examined the effects of dexamethasone on creatine kinase (CK) activity and insulin‐like growth factor I (IGF‐I) binding in two skeletal muscle‐derived cell lines (mouse, C2C12; rat, L6) and in one cardiac muscle‐derived cell line (rat, H9c2). Dexamethasone treatment during differentiation of cultured cells caused a dose‐dependent increase in CK activity as well as an increase in the degree of myotube formation in C2C12 and L6, whereas H9c2 cells did not exhibit significant CK activities during culture or dexamethasone treatment. Dexamethasone treatment of C2C12 did not stimulate proliferation in differentiating cultures, but a dose‐dependent increase in the number of nuclei was observed for L6 concomitant with increased CK activity. In L6 the increased CK activity may therefore reflect a dose‐dependent increase in proliferation. Short‐term (48 hr) treatment of C2C12 with dexamethasone (20 nM) did not appear to alter myoblast fusion but reversibly increased CK activity. In C2C12 the observed increase in CK, alanine aminotransferase (ALT), and aspartate aminotransferase (AST) activities with dexamethasone treatment suggest modulation of protein expression and/or turnover. Although the data for dexamethasone effects on CK activities varied in each of the cell lines, consistent behavior was observed in all three cell lines when IGF‐I binding was examined. IGF‐I binding to dexamethasone‐treated cells (50 nM for 24 hr the day prior to confluence) resulted in an increased number of available binding sites, with no effect on the binding affinities. Affinity cross linking and autoradiography indicated that the increase in IGF‐I binding was the result of dexamethasone up‐regulation of type I IGF receptors. Our data for all three muscle cell lines suggest that similar heterologous hormone receptor modulation of type I IGF receptor sites occurs with dexamethasone treatment.
Endogenous regulation of 2‐deoxyglucose uptake in c6 glioma cells correlates with cytoskeleton‐mediated changes of surface morphology
Authors: Lange Klaus , Brandt Ursulac , Keller Konrad , Zimmermann Bernd
Published: February 2005

The cellular basis of the membrane‐limited state of glucose utilization and the mechanism of the endogenous regulation of hexose uptake in dense monolayers of C6 glioma cells were investigated. In an earlier study, it was shown that at high rates of glucose transport and phosphorylation combined with the inhibition of glycolytic adenosine triphosphate (ATP) production by iodoacetate, an endogenous regulatory response occurred that resulted in rapid, periodic variations of the glucose uptake rates (Lange et al., 1982). Similar time‐dependent periodic changes of uptake rates also occurred during incubation of C6 glioma cells with 2 mM 2‐deoxyglucose (2‐DG) without pretreatment of the cells with iodoacetate. These changes were accompanied by variations of the intracellular ATP content, by distinct alterations of the shape and arrangement of microvilli and lamellae (lamellipodia) on the cell surface, and by changes of the cytoskeletal F‐actin content. Because the changes of 2‐DG uptake rates occurred independent of the intracellular 2‐DG concentration, the bulk of this 2‐DG pool was assumed to be localized apart from the membranal transport sites. Downregulation of 2‐DG uptake appeared to be triggered by a rapid decrease of a small pool of the cellular ATP involved in the phosphorylation of transported hexose. Scanning and transmission electron microscopic observations of cells fixed in different states of the endogenous uptake regulation supported the assumption that the interior of lamellae and microvilli may represent a small entrance compartment for transported hexoses in which occurred the observed close coupling between hexose transport and phosphorylation as well as the rapid variations of ATP content. Hexose uptake is supposed to be regulated by cytoskeleton‐mediated changes of volume and diffusional accessibility of this compartment, modulating the degree of its metabolic coupling with the cytoplasmic main compartment.
Differential effects of hyperoxia and hydrogen peroxide on DNA damage, polyadenosine diphosphate‐ribose polymerase activity, and nicotinamide adenine dinucleotide and adenosine triphosphate contents in cultured endothelial cells and fibroblasts
Authors: Junod Alain F. , Jornot Lan , Petersen Hilke
Published: February 2005

The effects of oxidative stress on DNA damage and associated reactions, increased polyadenosine diphosphate‐ribose polymerase (PARP) activity and decreased nicotinamide adenine dinucleotide (NAD) and adenosine triphosphate (ATP) contents, have been tested in primary cultures of porcine aortic endothelial cells. The cells were treated with 50‐500 μM H2O2 for 20 min or 100 μM paraquat for 3 days or were exposed to 95% O2 for 2 and 5 days. The administration of 250‐500 μM H2O2 resulted in a marked increase in PARP activity and a profound depletion of ATP and NAD. Although hyperoxia had no effect on PARP activity and reduced only slightly the ATP and NAD stores, it markedly reduced the ability of endothelial cells to increase PARP activity upon exposure to DNase. Paraquat had a similar effect. Human dermal fibroblasts were also exposed to 50‐500 μM H2O2 for 20 min or 95% O2 for 5 days. Their response to H2O2 differed from that of endothelial cells by their ability to maintain the ATP content at a normal level. Fibroblasts were also insensitive to the effect of hyperoxia. These results suggest that the oxidant‐related DNA damage is a function of the type of oxidative stress used and may be cell‐specific.
Diacylglycerol, but not inositol 1,4,5‐trisphosphate, accounts for platelet‐derived growth factor‐stimulated proliferation of BALB 3T3 cells
Authors: Suzuki‐Sekimori Reiko , Matuoka Koozi , Nagai Yoshitaka , Takenawa Tadaomi
Published: February 2005

Recently we found that an intracellular event related to phosphatidylinositol 4,5‐bisphosphate (PIP2) is crucial for platelet‐derived growth factor (PDGF)‐induced mitogenesis in fibroblastic cells (Matuoka, K., et al.: Science 239:640‐643, 1988). In the present study we examined the mitogenic effects of PIP2 and its hydrolysis products introduced into the cytoplasm of BALB 3T3 cells by micro‐injection to confirm the role of PIP2 hydrolysis in PDGF stimulation of cell proliferation. Injection of 1,2‐dioleylglycerol (diolein) into serum‐deprived quiescent cells induced DNA synthesis with the same time course as that induced by exposure of the cells to PDGF and, in the presence of PDGF, caused no additional increase in the cell population entering S phase. The injection of PIP2, inositol 1,4,5‐trisphosphate, or 1,2‐dioleylphosphatidic acid into the cells did not induce mitogenesis. Consistent results were obtained in experiments in which the cells were exposed to 1‐oleyl‐2‐acetylglycerol (OAG) and ionomycin; namely, OAG stimulated proliferation of BALB 3T3 cells, but ionomycin did not induce any mitogenesis. Desensitization of the protein kinase C pathway by prolonged exposure of the cells to phorbol ester abolished the induction of cell proliferation by subsequent injection of diolein or exposure to phorbol ester or OAG as well as by PDGF challenge. These findings strongly suggest that activation of the protein kinase C system following formation of diacylglycerol by PIP2 hydrolysis is mainly responsible for the mitogenic action of PDGF on BALB 3T3 cells.
Involvement and relative importance of at least two distinct mechanisms in the effects of 2‐mercaptoethanol on murine lymphocytes in culture
Authors: Pruett Stephen B. , Obiri Nicholas , Kiel Johnathan L.
Published: February 2005

2‐Mercaptoethanol (2‐ME) exerts several effects on murine lymphocytes in culture that might explain its ability to enhance survival and growth of these cells. The uptake of the essential amino acid cystine and consequently the maintenance of intracellular glutathione levels are enhanced by 2‐ME. Furthermore, 2‐ME (even in the disulfide form) causes lymphocytes to release thiols into the culture medium. These effects might protect the cells from oxidative damage. The additional cystine provided by treatment of lymphocyte cultures with 2‐ME might also allow adequate protein synthesis to support survival and/or growth. This study was conducted to assess the relative importance of the antioxidant and protein synthesis effects of 2‐ME. As expected, 2‐ME increased cystine uptake at all concentrations that enhanced growth and survival, but four nonthiol antioxidants that enhanced growth and/or survival either did not substantially affect cystine uptake or decreased it and did not affect the release of cystine or its products. The results presented here demonstrate that antioxidant protection is necessary and sufficient for lymphocyte survival and that cystine uptake in untreated lymphocytes is sufficient to support the protein synthesis needed for survival and limited growth. However, we also noted that concentrations of 2‐ME that stimulated maximal growth more than doubled protein synthesis as measured at 8 hr. Thus the portion of the effects of 2‐ME not accounted for by antioxidant action could be accounted for by enhanced protein synthesis.
Extracellular matrix: Differential influence on growth and biosynthesis patterns of vascular smooth muscle cells from SHR and WKY rats
Authors: Scott‐Burden Timothy , Resink Thérèse J. , Bürgin Maria , Bühler Fritz R.
Published: February 2005

Smooth muscle cells from spontaneously hypertensive rats (SHR) elaborated extracellular matrix (ECM) material in culture that was more stimulatory to growth of cells from normotensive (WKY) animals than their own matrix. Both cell types elaborated ECMs consisting of glycoconjugate material (proteoglycans, glycopeptides) elastin, and collagens, but there were differences in the relative proportions of the compounds synthesized. Cells from SHR produced an ECM richer in elastin than that synthesized by WKY derived cells (∼19% vs. 11%, respectively). However, the latter elaborated ECMs containing more (∼45% for WKY vs. 29% for SHR) glycoconjugate material than the former. The lysyloxidase‐mediated crosslinking of elastin was more rapid in cultured cells from SHR animals than from their normotensive counterparts and may be as a consequence of increased substrate (tropoelastin) availability in ECMs from SHR animals. The relative proportions and sulphate levels of the glycosaminoglycans associated with matrix material elaborated by the two cell types were similar. Radiolabelled glycoconjugate material was degraded by cells (SHR/WKY) when they were plated upon pre‐formed ECMs, and their patterns of synthesis of new matrix was markedly altered under such conditions. New matrix material elaborated by cells plated upon ECM‐coated dishes consisted predominantly of glycopeptide and proteoglycan matrix components. Epidermal growth factor promoted the incorporation of [3H]‐thymidine into DNA by quiescent cells, and this was also markedly stimulated when cells were plated onto ECM‐coated plasticware rather than onto plastic substratum.
Activation of the Na+/H+ exchanger by phorbol ester and osmotic shock is dependent on the degree of neutrophilic maturation
Authors: Costa‐Casnellie Maria R. , Cragoe E. J. , Segel George B. , Lichtman Marshall A.
Published: February 2005

Activation of neutrophils leading to superoxide production is accompanied by cytoplasmic alkalinization, which results from stimulation of the Na+/H+ exchanger. Since the exchanger undergoes permanent alterations during neutrophilic maturation of HL‐60 cells (Costa‐Casnellie et al.: journal of Biological Chemistry 263:11851–11855, 1988), we investigated whether its response to external stimuli such as phorbol esters or osmotic shock also was modified during cell maturation. Mature HL‐60 cells produce superoxide in response to active phorbol esters, whereas immature HL‐60 cells do not. Stimulation of the exchanger by active phorbol esters (phorbol 12‐myristate 13‐acetate or phorbol 12, 13‐dibutyrate) was observed in mature neutrophilic HL‐60 cells but not in their immature counterparts. Inactive 4‐alpha phorbol had no effect in either cell population. Compound H7 inhibited phorbol ester activation by 65%. In mature neutrophilic cells activation of the exchanger by phorbol esters caused two novel changes of its properties: (1) its apparent Km for Na+ transport increased 2‐fold; (2) its Vmax increased 2.6‐fold. Phorbol esters also caused a shift in pH dependence of activation similar to that induced in other cells. Osmotic shock, a different method known to activate the exchanger of other cells, induced activation in mature neutrophilic cells but not in immature cells. Thus, the response of the exchanger to external stimuli is affected by alterations occurring in association with cell maturation.
Transforming growth factor β‐1 positively modulates the bioactivity of fibroblast growth factor on corneal endothelial cells
Authors: Plouët Jean , Gospodarowicz Denis
Published: February 2005

Transforming growth factor beta‐1 (TGFβ‐1), known as an inhibitor of vascular endothelial cell proliferation in vitro, stimulates bovine corneal endothelial cells (BCE) proliferation. It also positively modulates the response of BCE cells to fibroblast growth factor (FGF) and epidermal growth factor (EGF). This effect is concentration dependent within a physiological range of TGFβ‐1, but it is blocked if cells are cultured on extracellular‐matrix‐coated dishes instead of plastic. TGFβ‐1 does not modify the number or the affinity of bFGF receptors on BCE cell surface but increases the bFGF content of these cells. This suggests that TGFβ‐1 might act through regulation of bFGF synthesis in BCE cells.
Basic fibroblast growth factor: Production, mitogenic response, and post‐receptor signal transduction in cultured normal and transformed fetal bovine aortic endothelial cells
Authors: Presta Marco , Maier Jeanette A. M. , Rusnati Marco , Ragnotti Giovanni
Published: February 2005

Normal FBAE AG 7680 cells and chemically transformed FBAE GM 7373 cells were compared for their capacity to produce and to respond to bFGF. Normal FBAE cells showed higher levels of bFGF protein and of poly(A)+ bFGF mRNA than transformed GM 7373 cells, indicating that chemical transformation in FBAE cells is paralleled by a decrease of bFGF gene expression. Basic FGF induced cell proliferation in both normal and transformed FBAE cells. However, bFGF appeared to be much more potent in transformed than in normal cells. No differences in bFGF membrane receptors were observed between normal and transformed FBAE cells in terms of apparent molecular weight, number per cell, dissociation constant, and kinetic of downregulation. In respect to normal cells, however, transformed GM 7373 cells showed higher basal levels of PKC activity. This kinase is activated by bFGF and is involved in mediating the mitogenic activity of bFGF, as shown by the capacity of the PKC inhibitor H‐7 to abolish the mitogenic activity of bFGF both in normal and transformed FBAE cells. Like bFGF, the PKC activators DAG and TPA exerted a stronger mitogenic activity in transformed than in normal FBAE cells. Thus, the different susceptibility of normal and transformed FBAE cells to bFGF appears to depend on differences in the post‐receptor signal transduction mediated by PKC rather than on differences in bFGF receptors.

The results indicate that chemical transformation causes significant modifications of bFGF physiology in FBAE cells. The relevance of these modifications to the genesis of tumors of vascular origin deserves further investigation.
Regulation of cerebroside sulfotransferase activity in cultured oligodendrocytes: Effect of growth factors and insulin
Authors: Fressinaud C. , Sarliève L. L. , Labourdette G.
Published: February 2005

Cerebroside sulfotransferase (EC 2.8.2.11, CST) specific activity has been determined in oligodendrocyte (OL)‐enriched glial cell cultures from newborn rat brain grown in serum supplemented medium. This activity is detectable at 5 days in vitro (DIV) and reaches its maximum value at 12 DIV. This period corresponds to that of oligodendrocyte precursor proliferation in these cultures. The activity decreases thereafter and remains nearly constant after 24 DIV. The developmental curve of CST activity is parallel in pure oligodendrocyte subcultures but twice higher than in primary cultures. These data confirm that CST is highly enriched in OL. Basic fibroblast growth factor (bFGF) (15 ng/ml) and platelet derived growth factor (PDGF) (0.75 U/ml) both enhance CST activity by 90% and 72%, respectively. This increase is in the same range than that of DNA content in treated cultures, whereas protein increase is smaller (50% and 22%, respectively). In contrast, transforming growth factor β1 (TGFβ1, 0.5 and 5 ng/ml) does not significantly enhance CST activity nor DNA content of OL cultures. Insulin at high concentrations (5 μg/ml) also enhances CST activity but has no effect at physiological concentrations (20 ng/ml). These results show that CST activity can be controlled by growth factors. They suggest that CST activity is more closely related to OL and OL precursor proliferation than to myelination itself since its maximal activity preceeds myelination in vitro.
Forskolin has biphasic effects on osteoprogenitor cell differentiation in vitro
Authors: Turksen Kursad , Grigoriadis Agamemnon E. , Heersche Johan N. M. , Aubin Jane E.
Published: February 2005

Cells isolated from fetal rat calvaria (RC) and maintained in vitro in medium containing ascorbic acid and B‐glycerophosphate form three‐dimensional, mineralized nodules having the histological, immunohistological, and ultrastructural characteristics of woven bone. We have studied the effects of forskolin (FSK), a diterpene that activates adenylate cyclase, in this system. While 10−7 −10−5 M FSK significantly stimulated cAMP levels in RC cells, lower concentrations did not. cAMP levels with 10−5 M FSK reached a maximum by 30 min at 37°C and returned to basal level in 2‐3 hr. Changes in cAMP levels correlated with changes in cellular shape: cells treated with 10−5 M FSK assumed a stellate morphology, lost microfilament bundles, and reduced their substrate adhesiveness, while cells treated with 10−9 M were not affected. Exponential growth and saturation densities of FSK‐treated cultures were similar to untreated cultures, indicating that FSK was neither toxic nor stimulatory to the population. The effect on bone nodule formation of FSK present continuously depended on concentration: 10−5 M FSK significantly inhibited the number of nodules formed, while 10−9 M FSK significantly stimulated bone nodule formation. Single short treatments with either 10−5 M or 10−9 M FSK had no effect on nodule formation, but repeated short duration treatments (1 hr every 2 days for 21 days) gave results similar to continuous exposure. These results indicate that intermittent elevations in intracellular cAMP have an inhibitory effect on bone formation. In addition, our work indicates that low concentrations of FSK stimulate differentiation of osteoprogenitor cells possibly through a non‐cAMP‐dependent process.
Cyclic adenosine monophosphate levels and the function of skin microvascular endothelial cells
Authors: Tuder Rubin M. , Karasek Marvin A. , Bensch Klaus G.
Published: February 2005

The maintenance of the normal epithelioid morphology of human dermal microvascular endothelial cells (MEC) grown in vitro depends strongly on the presence of factors that increase intracellular levels of cyclic AMP. Complete removal of dibutyryl cAMP and isobutylmethyixanthine (IMX) from the growth medium results in a progressive transition from an epithelioid to a spindle‐shaped cell line. This transition cannot be reversed by the readdition of dibutyryl cAMP and IMX to the growth medium or by addition of agonists that increase cAMP levels. Spindle‐shaped MEC lose the ability to express Factor VIII rAG and DR antigens and to bind peripheral blood mononuclear leukocyte (PBML).

Ultrastructural analyses of transitional cells and spindle‐shaped cells show decreased numbers of Weibel‐Palade bodies in transitional cells and their complete absence in spindle‐shaped cells. Interferon‐γ alters several functional properties of both epitheliod and spindle‐shaped cells. In the absence of dibutyryl cAMP it accelerates the transition from epithelial to spindle‐shaped cells, whereas in the presence of cyclic AMP interferon‐γ increases the binding of PBMLs to both epithelioid and spindle‐shaped MEC and the endocytic activity of the endothelial cells.

These results suggest that cyclic AMP is an important second messenger in the maintenance of several key functions of microvascular endothelial cells. Factors that influence the levels of this messenger in vivo can be expected to influence the angiogenic and immunologic functions of the microvasculature.
Effects of reduced cell volume and water content on glycolysis in L‐929 cells
Authors: Clegg James S. , Jackson Susan A. , Fendl Karin
Published: February 2005

Mouse L‐929 cells were subjected to increasing concentrations of sorbitol, which remove cell water and reduce volume osmotically. The rate of lactate production from glucose was significantly higher in osmotically perturbed cells than in controls, both in monolayers and in suspensions. L cells can apparently use sorbitol as a glycolytic substrate; however, studies using other solutes (trehalose and sucrose) and permeabilized cells showed that the major effect of scrbitol on glycolysis in intact cells is mediated through a reduction in cell water content and volume. It is possible to explain some of these results by an increase in the chemical potentials of dissolved components of the glycolytic pathway caused by water loss; however, the relationship between water loss and glycolytic rate increase in not a simple linear one, suggesting that the situation is more complex than would result merely from increased concentrations of pathway components. Whatever the complete explanation might be, these studies show that glycolysis continues in an orderly fashion in cells that have lost about 85% of their original water content, suggesting that the operation of this pathway is not unduly sensitive to events taking place in the bulk aqueous phase.
Correlation between fibronectin and its receptor in chick myoblast differentiation
Authors: Chung Chang Yong , Kang Man‐Sik
Published: February 2005

Alterations in the amount of fibronectin and in the number of its receptors during myoblast differentiation of chicken embryo were investigated. The amount of fibronectin in the cell surface pool as measured by immunoblotting decreased during myogenesis To identify and characterize the fibronectin receptors on the myoblasts, the interactions of the 28,000 dalton (28 kDa) amino terminal fragment and 85,000 dalton (85 kDa) cell‐binding fragment of fibronectin with my‐oblasts were examined. The binding of the 28 kDa fragment was found to be time‐dependent and reached a maximum level within 60 min. The unlabeled 28 kDa fragment inhibited the binding of the radioiodinated 28 kDa fragment, whereas the unlabeled 85 kDa fragment and antibody to integrin did not inhibit it, suggesting that the 28 kDa fragment interacts with the matrix assembly receptors but not with the cell adhesion receptors. There was a single class of 3.4 × 105 binding sites per cell with an apparent dissociation constant of 1.4 × 10−7 M on 30 hr old myoblasts. The specific binding of the radioiodinated 28 kDa fragment to myoblasts decreased as the fusion proceeded. This decrease of binding was consistent with the decrease in the amount of fibronectin. Furthermore, the levels of fibronectin and binding of the radioiodinated 28 kDa fragment in the fusion‐blocked myoblasts by EGTA treatment appeared to remain constant. These results suggest that the decrease and/or loss of fibronectin during myoblast fusion is closely correlated with the alteration of fibronectin receptors and with the fusion of myoblasts.
Menthol blocks dihydropyridine‐insensitive Ca2+ channels and induces neurite outgrowth in human neuroblastoma cells
Authors: Sidell Neil , Verity M. Anthony , Nord Edward P.
Published: February 2005

Voltage‐gated Ca2+ channels were identified in LA‐N‐5 human neuroblastoma cells using the Ca2+ sensitive fluorescent probe, fura‐2. Using a variety of “classical” Ca2+ channel blockers, we have demonstrated the presence of both dihydropyridine (DHP)‐sensitive and ‐insensitive channel types that can be activated by depolarization of the cells with either high K+ or gramicidin in the bathing solution. Brief exposure of LA‐N‐5 cells to menthol blunted the depolarization‐induced Ca2+ influx though both DHP‐sensitive and DHP‐insensitive channels. This effect is concentration dependent (50% maximal blocking effect with 0.25 mM menthol), rapid in onset, and readily reversible. The specificity of the Ca2+ ‐channel blocking effect of menthol was demonstrated in parallel studies using compounds with similar structures: menthone blocked Ca2+ channels with about half the potency of menthol, while cyclohexanol was without effect. Addition of either menthol or menthone to LA‐N‐5 cultures induced neurite outgrowth, cellular clustering, and reduction of cell growth in a dose‐dependent fashion that correlated with the ability of these compounds to inhibit the DHP‐insensitive Ca2+ influx. Cyclohexanol had no biologic activity. Taken together, the parallel potency for blockade of DHP‐insensitive Ca2+ influx with the biologic activity of menthol suggests a role for certain types of Ca2+ channels in triggering growth and morphologic changes in LA‐N‐5 cells.
Novel monoclonal antibodies identify antigenic determinants unique to cellular senescence
Authors: Porter Mary Beth , Pereira‐Smith Olivia M. , Smith James R.
Published: February 2005

Normal human diploid fibroblasts exhibit a limited lifespan in vitro and are used as a model to study in vivo aging. Monoclonal antibodies were generated against partially purified surface membranes from human diploid fibroblasts at the end of their lifespan (senescent). Three hybridomas were isolated that secreted antibodies reacting to cellular determinants expressed specifically on senescent human fibroblasts of different origin, including neonatal foreskin, embryonic lung, and adult skin punch biopsy, but not expressed on matched young cells. The antibodies did not bind to immortal human cells and normal young cells made reversibly nondividing, indicating the antigens are not expressed in cells that are not senescent. The antibodies identified senescent cells in a mixed cell population and expression of the senescent cell antigens correlated strongly with the cells inability to synthesize DNA at the onset of senescence. The antigens appeared to be cell surface or extracellular matrix associated, and the epitopes were destroyed by mild trypsin treatment. Western analysis indicated all three antibodies reacted with fibronectin. Though the antigenic determinants on the fibronectir molecule were not accessible in the intact young cell, the epitopes were present in fibronectin extracted from both senescent and young cells, as well as purified human plasma fibronectin. These antibodies and the senescent specific expression of the antigens provide powerful tools to investigate the mechanisms leading to in vitro senescence. This may enable us to investigate directly the relationship between cellular aging and aging of the individual.
Macrophage‐colony‐stimulating factor (CSF‐1) modulates a differentiation‐specific inward‐rectifying potassium current in human leukemic (HL‐60) cells
Authors: Wieland Steven J. , Chou Robin H. , Gong Qi‐Hua
Published: February 2005

A voltage‐activated inward‐rectifying K+ conductance (Iki) appears in human promyelocytic leukemia (HL‐60) cells during phorbol ester‐induced differentiation into macrophages. This conductance was detected in the cells 24 hours after exposure to phorbol‐12‐myristate‐13‐acetate (PMA), as the cells began to express the macrophage phenotype, and continued to increase for 4 days after PMA exposure. The magnitude of inward current was a function of external K+; current was blocked by extracellular or intracellular Cs+ and by extracellular Ba++. Hyperpolarization produced activation at membrane potentials more negative than −80 mV, and a slower, partial inactivation also occurred at potentials more negative than −100 mV. This conductance was not detected in proliferating cells nor in granulocytes derived from HL‐60 cells which were induced to differentiate with retinoic acid (RA). Exposure of differentiated macrophages to recombinant human CSF‐1 produced inhibition of the Iki beginning within 1 minute after exposure. CSF‐1 inhibition of Iki channels in cell‐attached patches indicated that channel modulation was via intracellular mediators. The rapid inhibition of the inward rectifier by the macrophage‐specific CSF‐1 appears to be one of the earliest cellular responses to this factor.
Fluid shear stress as a mediator of osteoblast cyclic adenosine monophosphate production
Authors: Reich Kathleen M. , Gay Carol V. , Frangos John A.
Published: February 2005

Effects of interstitial fluid flow on osteoblasts were investigated. Intracellular cyclic adenosine monophosphate (cAMP) levels were monitored in cultured osteo‐blasts subjected to shear rates ranging from 10 to 3,500 sec−1. Cyclic AMP levels were significantly increased at all shear rates from 1 pmole/mg protein to 10‐16 pmole/mg protein. Osteoblasts subjected to a shear rate of 430 sec−1 for 0.5‐15 minutes exhibited elevated levels (12‐fold) of intracellular cAMP, which were sustained throughout the perfusion period. Osteoblasts were three times more sensitive to flow stimulation than human umbilical vein endothelial cells and baby hamster kidney fibroblasts, which also displayed higher cAMP levels (4‐fold) after exposure to flow. To distinguish streaming potential effects from shear stress effects, viscosity was increased 5‐fold by addition of neutral dextran to the perfusing medium. Shear stress is a function of viscosity, and streaming potentials are not for a given shear rate. The mechanism of this cellular response to flow was shown to be shear stress dependent. Inhibition of cyclooxygenase by 20 μM ibuprofen completely inhibited the flow‐dependent cAMP response, indicating the cAMP response is mediated by prostaglandins. Our results suggest that fluid flow induced by mechanical stress may be an important mediator of bone remodeling.
Kinetics of hydrolysis of endocytosed substrates by mammalian cultured cells: Early introduction of lysosomal enzymes into the endocytic pathway
Authors: Bowser Robert , Murphy Robert F.
Published: February 2005

The kinetics of exposure of endocytosed material to two lysosomal enzymes were determined for a number of cultured cell lines using fluorogenic substrates. Hydrolysis of endocytosed substrates for cathepsin B and acid phosphatase was observed to begin within 3–10 min of substrate addition and to proceed linearly for up to 60 min thereafter. Hydrolysis of the cathepsin B substrate was not affected by inhibition of protein synthesis with cycloheximide, indicating that the enzymes present in early endosomes are not exclusively newly synthesized. As had been observed previously for a cathepsin B substrate (Roederer, M., Bowser, R., and Murphy, R.F., J. Cell. Physiol., 131:200–209, 1987), hydrolysis of the acid phosphatase substrate was not blocked at temperatures below 20°C. The results suggest that the endosome is the primary site of initial exposure of endocytosed material to hydrolytic enzymes.
Expression of simple epithelial cytokeratins in bovine pulmonary microvascular endothelial cells
Authors: Patton Wayne F. , Yoon Min Ung , Alexander J. Steven , Chung‐Welch Nancy , Hechtman Herbert B. , Shepro David
Published: February 2005

Polypeptides of bovine aortic, pulmonary artery, and pulmonary microvascular endothelial cells, as well as vascular smooth muscle cells and retinal pericytes were evaluated by two‐dimensional gel electrophoresis. The principal cytoskeletal proteins in all of these cell types were actin, vimentin, tropomyosin, and tubulin. Cultured pulmonary microvascular endothelial cells also expressed 12 unique polypeptides including a 41 kd acidic type 1 and two isoforms of a 52 kd basic type II simple epithelial cytokeratin. Pulmonary microvascular endothelial cell expression of the simple epithelial cytokeratins was maintained in culture in the presence or absence of retinal‐derived growth factor, and regardless of whether cells were cultured on gelatin, fibronectin, collagen I, collagen IV, laminin, basement membrane proteins, or plastic. Cytokeratin expression was maintained through at least 50 population doublings in culture. The expression of cytokeratins was found to be regulated by cell density. Pulmonary microvascular endothelial cells seeded at 2.5 × 105 cells/cm2 (confluent seeding) expressed 3.5 times more cytokeratins than cells seeded at 1.25 × 104 cells/cm2 (sparse seeding). Vimentin expression was not altered by cell density. By indirect immunofluorescence microscopy it was determined that the cytokeratins were distributed cytoplasmically at subconfluent cell densities but that cytokeratin 19 sometimes localized at regions of cell‐cell contact after cells reached confluence. Vimentin had a cytoplasmic distribution regardless of cell density. These results suggest that pulmonary microvascular endothelial cells have a distinctive cytoskeleton that may provide them with functionally unique properties when compared with endothelial cells derived from the macrovasculature. In conjunction with conventional endothelial cell markers, the presence of simple epithelial cytokeratins may be an important biochemical criterion for identifying pulmonary microvascular endothelial cells.
Adenosine metabolism in kidney slices under normoxic conditions
Authors: Blanco Julià , Mallol Josefa , Lluis Carmen , Canela Enric I. , Franco R.
Published: February 2005

The effect of adenosine in rat kidney under normoxic conditions has been studied. It is demonstrated that adenosine modulates cell nucleotide levels. HPLC analysis of the purine compounds inside the cell indicates that adenosine improves the ATP/ADP ratio, whereas it diminishes the adenine content. This behaviour is not due to mediation by specific receptors, as agonists at P1 purinoceptors did not have any effect. Further evidence using inosine as well as dipiridamole and deoxycoformycin indicates that all effects are dependent on the previous uptake of adenosine. The origin of free adenine in the kidney has been investigated, and it appears to come from the phosphorolysis of 5′‐methylthio‐adenosine. This report is the first to describe the activity of methylthioadenosine phosphorylase (E.C. 2.4.2.28) in the kidney. It is concluded that 1) extracellular adenosine improves guinea pig renal function by increasing the ATP level and the ATP/ADP ratio; and 2) there exists a functional pathway in the kidney that produces adenine and AMP coming from methionine and ATP. This latter pathway probably produces spermine and spermidine, which are likely to be important for renal function.
Effect of transforming growth factor beta on proliferation of L6 and embryonic porcine myogenic cells
Authors: Pampusch Mary S. , Hembree Joan R. , Hathaway Marcia R. , Dayton William R.
Published: February 2005

We have examined the effect of T‐ansforming Growth Factor (TGF) beta on proliferation of L6 and embryonic porcine myogenic cells. Proliferation of L6 cells was suppressed by both TGF beta‐1 and TGF beta‐2 in a dose‐dependent manner. Half‐maximal suppression of proliferation occurred at.036 ng TGF beta‐1/ml and.06 ng TGF beta‐2/ml. Maximal inhibition (60% suppression of proliferation for TGF beta‐1 and 52% for TGF beta‐2) occurred between.1 and.3 ng/ml for each growth factor. Suppression of proliferation was completely abolished in the presence of an anti‐TGF beta antibody that inhibited the biological activity of TGF beta‐1 and TGF beta‐2. When we evaluated the effect of TGF beta‐1 on proliferation of embryonic porcine myogenic cells we obtained results which were very similar to those obtained for L6 cells. Insulin‐like growth factor (IFG)‐I stimulated proliferation of L6 cells in a dose‐dependent manner in serum‐free, defined medium. However as little as.02 ng TGF beta‐1/ml detectably suppressed this stimulation and.3 ng TGF beta‐1/ml caused a 60% reduction in cell number in cultures treated with 30 ng IGF‐1/ml. Thus TGF beta‐1 significantly suppressed IGF‐l‐stimulated proliferation of L6 cells.
Effect of epidermal growth factor on the expression of protooncogenes c‐myc and c‐Ha‐ras in short‐term primary hepatocyte culture
Authors: Kost Diane P. , Michalopoulos George K.
Published: February 2005

We have characterized the effect of the hepatomitogen epidermal growth factor (EGF) on the expression of the cellular protooncogenes c‐Ha‐ras and c‐myc in short‐term (48 hours) primary hepatocyte culture. mRNA concentrations of both protooncogenes increased dramatically in nonproliferating cultures and in the absence of EGF, suggesting that the isolation procedure or the culture conditions may trigger expression of these genes or potentially increase the lifetime of transcripts in vitro, regardless of the presence of a mitogen. In cells treated with EGF, a distinct peak in c‐Ha‐ras expression was seen 24 hours after EGF treatment. This coincided with the onset of DNA synthesis. No such peak was seen in cultures not treated with EGF. The c‐myc mRNA concentrations were increased relatively equally in all cultures with or without the addition of EGF. These data show a differential response of these two cell‐cycle‐associated genes to the culture conditions and EGF stimulation. It also demonstrated that enhanced gene expression for Ha‐ras and myc in hepatocytes can occur in the absence of cell proliferation.