Decreased vascular glucose transporter expression and glucose uptake in DOCA-salt hypertension.

OBJECTIVE: Because glucose uptake and metabolism can affect vascular smooth muscle cell function, we proposed that animals with hypertension might develop alterations in glucose transporter expression in vascular smooth muscle cells that were responsible for some of the vascular abnormalities characteristic of hypertension. DESIGN AND METHOD: Male Sprague-Dawley rats (250-300 g) were left uni-nephrectomized and either implanted or not with deoxycorticosterone acetate (DOCA, 200 mg/kg) impregnated silastic. All animals were fed normal rat chow. The DOCA-implanted rats were given water supplemented to 1% NaCl and 0.2% KCl for 7, 14 or 28 days. RESULTS: The insulin-response glucose transporter (GLUT4) polypeptide levels were depressed several-fold in aortae and carotid arteries from DOCA-salt hypertensive rats compared with sham rats. Uptake of the glucose analog, 2-deoxyglucose (2-DOG), was also reduced 53% in hypertensive compared with sham aortae. There were no changes in GLUT4 expression in other tissues in the DOCA-salt animals, nor were there significant changes in aortae from spontaneously hypertensive rat/stroke prone animals. As previously demonstrated, carotid arteries from DOCA-salt animals exhibited a significant increased contractile sensitivity to ergonovine. Inhibition of glucose metabolism with 2-DOG in sham arteries caused a marked enhancement of contractile responsiveness to ergonovine, whereas 2-DOG had no effect on the already enhanced contractility of DOCA-salt arteries, suggesting that reduction in glucose uptake and metabolism substantially increases the contractile response of DOCA-salt arteries. CONCLUSIONS: Alterations in glucose uptake and metabolism in vascular smooth muscle cells may participate in the contractile abnormalities characteristic of certain forms of hypertension.

Chelerythrine chloride induces rapid polymorphonuclear leukocyte apoptosis through activation of caspase-3.

Polymorphonuclear leukocytes (PMN) play a primary role in the initiation and propagation of inflammatory responses. PMN apoptosis is a major mechanism associated with the resolution of inflammatory reactions. Understanding mechanisms associated with PMN apoptosis will be of critical value in the development of novel pharmacological treatment strategies for local and/or systemic inflammatory disorders. The present study demonstrates that chelerythrine chloride induces human PMN to undergo rapid and synchronous progression into the apoptotic process via a PKC-independent mechanism. The appearance of the morphological features of apoptosis in chelerythrine-treated PMN is preceded by a significant upregulation in caspase-3 activity. GM-CSF (a cytokine that protects PMN in several models of PMN apoptosis) does not protect PMN from chelerythrine chloride-induced apoptosis.

N-acetylcysteine and endothelial cell injury by sulfur mustard.

Understanding the underlying mechanisms of cell injury and death induced by the chemical warfare vesicant sulfur mustard (HD) will be extremely helpful in the development of effective countermeasures to this weapon of terror. We have found recently that HD induces both apoptosis and necrosis in endothelial cells (Toxicol. Appl. Pharmacol. 1996; 141: 568-583). Pretreatment of the endothelial cells for 20 h with the redox-active agent N-acetyl-L-cysteine (NAC) selectively prevented apoptotic death induced by HD. In this study, we tested the hypotheses that pretreatment with NAC acts through two different pathways to minimize endothelial injury by HD: NAC pretreatment acts via a glutathione (GSH)-dependent pathway; and NAC pretreatment acts to suppress HD-induced activation of the nuclear transcription factor NFkappaB. We used a fluorescence microscopic assay of apoptotic nuclear features to assess viability and electrophoretic mobility shift assays (EMSAs) to assess the activity of NFkappaB following exposure to HD. The cells were treated with 0-10 mM GSH for 1 h prior to and during exposure to 0 or 500 microM HD for 5-6 h. Cells were also treated with 50 mM NAC or 200 microM buthionine sulfoximine (BSO), an inhibitor of GSH synthesis, alone or in combination overnight prior to exposure to 0 or 500 microM HD for 5-6 h. Externally applied GSH up to a concentration of 5 mM had no toxic effect on the cells. Mild toxicity was associated with 10 mM GSH alone. There was a dose-related enhancement of viability when 2.5 and 5 mM GSH were present during the HD exposure. Pretreatment with BSO alone had no discernible toxicity. However, pretreatment with this inhibitor of GSH synthesis potentiated the toxicity of HD. Pretreatment with 50 mM NAC, as previously reported, provided substantial protection. Combining pretreatment with both BSO and NAC eliminated the protective effect of NAC pretreatment alone on HD injury. These observations are highly suggestive that NAC enhances endothelial survival via GSH-dependent effects and confirms and extends the work of others with different models that externally supplied GSH alone may be a fairly effective countermeasure against HD injury of endothelium. We next examined the hypothesis that HD may activate the nuclear transcription factor NFkappaB by performing EMSAs with nuclear extracts of endothelial cells following exposure to 0, 250 or 500 microM HD. This demonstrated an up to 2.5-fold increase (scanning densitometry) in activation of NFkappaB binding to its consensus sequence induced by 500 microM HD after 5 h of HD exposure. Paradoxically, treatment of the endothelial cells alone with 50 mM NAC activated NFkappaB, although HD-induced activation of NFkappaB was partially suppressed by NAC at 5 h. Factor NFkappaB is an important transcription factor for a number of cytokine genes (e.g. tumor necrosis factor, TNF), which can be activated following stress in endothelial cells. Taken together, these observations suggest that the protective effects of NAC may be mediated by enhanced GSH synthesis. The increased GSH may act to scavenge HD and also prevent oxidative activation of NFkappaB. Under some conditions, NAC may act as an oxidizing agent and thus increase NFkappaB activity. The NFkappaB-dependent gene expression may be important in inducing endothelial cell death as well as in generating a local inflammatory reaction associated with the release of endothelial-derived cytokines.

Activation of poly [ADP-Ribose] polymerase in endothelial cells and keratinocytes: role in an in vitro model of sulfur mustard-mediated vesication.

Although endothelial cells and keratinocytes appear to be the primary cellular targets of sulfur mustard (SM), the role of the nuclear enzyme poly (ADP-ribose) polymerase (PARP) in SM-induced vesication has not been clearly defined. PARP is thought to play a crucial role in DNA repair mechanisms following exposure to alkylating agents like SM. Using a combination of fluorescence microscopy and biochemical assays, we tested the hypothesis that SM causes activation of PARP in endothelial cells and keratinocytes with subsequent loss of nicotinamide adenine dinucleotide (NAD) and depletion of adenosine triphosphate (ATP) levels. To determine if PARP activation accounts for SM-induced vesication, keratinocyte adherence and permeability of endothelial monolayers were measured as in vitro correlates of vesication. As early as 2 to 3 h after exposure to SM concentrations as low as 250 microM, dramatic changes were induced in keratinocyte morphology and microfilament architecture. Exposure to 500 microM SM induced a fourfold increase in PARP activity in endothelial cells, and a two- to threefold increase in keratinocytes. SM induced a dose-related loss of NAD+ in both endothelial cells and keratinocytes. ATP levels fell to approximately 50% of control levels in response to SM concentrations >/=500 microM. SM concentrations >/=250 microM significantly reduced keratinocyte adherence as early as 3 h after exposure. Endothelial monolayer permeability increased substantially with concentrations of SM >250 microM. These observations support the hypothesis that the pathogenic events necessary for SM-induced vesication (i.e., capillary leak and loss of keratinocyte adherence) at higher vesicating doses of SM (>/=500 microM) may depend on NAD loss with PARP activation and subsequent ATP-dependent effects on microfilament architecture. Vesication developing as a result of exposure to lower concentrations of SM presumably occurs by mechanisms that do not depend on loss of cellular ATP (e.g., apoptosis and direct SM-mediated damage to integrins and the basement membrane).

Protein extracts of dentin affect proliferation and differentiation of osteoprogenitor cells in vitro.

Proteins associated with the mineral phase of dentin are considered to have the potential to alter cell function within the local environment, during development and regeneration of tooth/periodontal tissues. Cells that may be altered include osteoblasts, ameloblasts, periodontal ligament cells, odontoblasts, and cementoblasts. However, specific factors within dentin controlling cell activity have not been elucidated. To investigate further the role of dentin proteins in regulating cell behavior, MC3T3-E1 cells, a mouse osteoprogenitor cell line, were exposed to guanidine/EDTA extracts of dentin (G/E-D) prepared from bovine teeth. Cells, with or without G/E-D (2 to 50 microg/ml), were evaluated for proliferative activity and for mRNA expression of bone-associated genes. Results indicated that G/E-D suppressed cell proliferation and caused striking morphological changes, including the conversion of cuboidal cells into fibroblastic, spindle-shaped cells. Markers of osteoblast differentiation, osteocalcin and bone sialoprotein mRNA were decreased, while osteopontin mRNA was enhanced in cells exposed to G/E-D. Since transforming growth factor beta (TGFbeta1) has been reported to influence cells in a similar fashion, G/E-D were examined for the presence of and concentration of TGFbeta using slot blot analysis and enzyme immunoassay (ELISA), respectively. These analyses demonstrated that G/E-D contained 6.6 ng/mg of TGFbeta1. Next, cells were exposed to G/E-D in conjunction with anti-TGFbeta1,2,3 antibody. When cells were exposed to antibody, G/E-D-mediated changes in morphology and gene expression were blocked. These results suggest that TGFbeta1 and perhaps other factors in dentin can regulate cell behavior and, therefore, can influence development, remodeling, and regeneration of mineralized tissues.

Stimulation of osteopontin mRNA expression in HL-60 cells is independent of differentiation.

12-O-Tetradecanoylphorbol 13-acetate (TPA) induces HL-60 cells to differentiate along the monocyte/macrophage pathway and stimulates expression of the extracellular adhesion protein osteopontin (OPN). In this study, the mechanism of TPA-mediated OPN mRNA expression and its relationship to differentiation were investigated. The induction of OPN mRNA by TPA was dose dependently inhibited by staurosporine (0.4-10.0 nM) and chelerythrine (0.1-5.0 microM), indicating that OPN expression requires PKC activation. Furthermore, the mitogen-activated protein kinase kinase (MAPKK) inhibitor, PD 098059 (1.0-10.0 microM), inhibited the effect of TPA in a dose-dependent fashion. Cycloheximide (10 microg/ml) ablated the induction of OPN mRNA by TPA. To determine if OPN mRNA expression was associated with a particular differentiational pathway, HL-60 cells were treated with RA, 9-cis-RA, calcitriol, or sodium butyrate. None of these agents stimulated OPN mRNA. Treatment with TPA subsequent to a 120-h pretreatment with retinoic acid (RA), 9-cis-RA, or calcitriol resulted in a potentiation of the induction of OPN mRNA. These results support a role for protein kinase C (PKC) in promoting OPN expression because each of these agents increased PKC levels. An hOPN promoter/reporter construct was responsive to TPA, indicating that this effect is at the level of transcription. Thus, TPA-stimulated transcription of the OPN gene apparently occurs via a PKC/MAPK-dependent mechanism that is independent of that associated with differentiation and is not dependent on the maturational state of these cells.

Comparative responsiveness of HL-60, HL-60R, and HL-60R+ (LRARSN) cells to retinoic acid, calcitriol, 9 cis-retinoic acid, and sodium butyrate.

In HL-60 cells, retinoic acid (RA) and 9 cis-RA induce granulocytic differentiation, and calcitriol and sodium butyrate induce monocytic differentiation. To study the role of retinoid resistance on the response to these agents, we investigated their effects in HL-60 cells, retinoid-resistant HL-60R cells, and HL-60R+ cells in which retinoid sensitivity has been restored. In HL-60 cells, cathepsin D (ctsd) mRNA levels are increased by these agents and by cholera toxin after pretreatment with each agent. Calcitriol, 9 cis-RA, and sodium butyrate increase interleukin-8 (IL-8) mRNA expression, and pretreatment with these agents or RA potentiates the stimulation of IL-8 by phorbol ester (TPA). Pretreatment of HL-60 cells with all of the agents confers inducibility of cathepsin L (ctsl) mRNA by TPA in previously unresponsive cells. In HL-60R cells, none of the agents alone or in combination significantly enhances the expression of the ctsd, IL-8, or ctsl mRNAs. Retinoid stimulation (either alone or in combination with the other agents) of the three mRNAs is partially restored in the HL-60R+ cells. Calcitriol does not alter the expression of any of these mRNAs, and only the stimulation of IL-8 mRNA by sodium butyrate is recovered. Treatment with all of the agents inhibits proliferation and stimulates differentiation of the HL-60 cells. RA and calcitriol are unable to inhibit proliferation of the HL-60R cells, whereas only calcitriol fails to inhibit proliferation of the HL-60R+ cells. None of the agents induces differentiation in either the HL-60R or HL-60R+ cells. Therefore, the mutation of the RA receptor alpha is insufficient to account for the altered responses of the HL-60R cells, and there are likely defects in other signaling pathways in these cells. These cells may prove useful in examining the mechanism of cross-resistance between various differentiating agents.

Effect of 1,10-phenanthroline on DNA binding by 1,25-(OH)2 vitamin D receptor.

The effect of the chelator 1,10-phenanthroline on the DNA-binding of 1,25-dihydroxyvitamin D receptor was demonstrated. Increasing concentrations of the chelator, up to 3 mM, decreased receptor binding to DNA-cellulose. This effect was specific for the chelating function because the nonchelating analog, 1,7-phenanthroline, did not inhibit DNA-cellulose binding of the receptor. The cations Cd2+ and Zn2+ were able to block chelator-mediated inhibition. These cations were unable to reverse the inhibition unless 1,10-phenanthroline was removed by dialysis.

Regulation of cathepsin D gene expression in HL-60 cells by retinoic acid and calcitriol.

Cathepsin D (ctsd) is a lysosomal acid protease found in neutrophils and monocytes. We investigated whether differentiating agents increase the expression of ctsd mRNA in HL-60 cells. Treatment with either retinoic acid or calcitriol enhances the steady-state levels of ctsd mRNA in a dose-dependent manner. The stimulation by retinoic acid requires new protein synthesis. Pretreatment with retinoic acid enhances the response of the ctsd gene to prostaglandin E2. To determine whether the effects of retinoic acid and calcitriol are associated with differentiation, we pretreated Hl-60 cells for 120 h with inducers of granulocytic differentiation (lithium chloride, DMSO, and retinoic acid) and monocytic differentiation (calcitriol, sodium butyrate, and phorbol ester). Lithium chloride and DMSO do not significantly affect ctsd mRNA expression, and none of the granulocytic inducers alters the subsequent response of the ctsd gene to calcitriol. All of the monocytic inducers stimulate ctsd mRNA, and both calcitriol and sodium butyrate significantly potentiate the subsequent response to retinoic acid. Transcription initiation of the ctsd gene occurs at one major and several minor sites and is unaffected by treatment with retinoic acid and calcitriol or pretreatment with other differentiating agents. Although differentiation appears to influence ctsd mRNA expression, calcitriol and retinoic acid stimulate ctsd gene expression via mechanisms that are independent of their role in differentiation.

Phorbol ester stimulated cathepsin L expression in U937 cells.

Cathepsin L (ctsl) is a lysosomal cysteine proteinase, the synthesis and secretion of which is induced by transformation, growth factors, and tumor promoters. We studied the effect and the mechanism of action of phorbol ester (TPA) on the expression of ctsl mRNA in U937 histiocytic leukemia cells. TPA treatment induces ctsl mRNA in a manner that is dose-dependent, occurs at the level of transcription, and is ablated by cotreatment with cycloheximide but is unaffected by dexamethasone. Treatment with TPA plus staurosporine, a potent protein kinase C inhibitor, results in greater expression of ctsl mRNA than does treatment with TPA alone. Similar to TPA, staurosporine alone increases ctsl transcription, an effect that is inhibited by cycloheximide. Another PKC inhibitor, H7, exerted no effect upon the induction of ctsl mRNA by either TPA or staurosporine. Staurosporine and H7, however, inhibit the increase in c-jun mRNA by TPA. In contrast, the tyrosine kinase inhibitors herbimycin A and genistein inhibit the effect of TPA and staurosporine upon ctsl mRNA with little or no effect on c-jun expression. Pretreatment with sodium orthovanadate enhances the induction of ctsl expression by TPA and staurosporine. These data suggest that, in U937 cells, TPA-stimulated ctsl gene transcription is apparently activated by a protein kinase C-independent signal transduction pathway involving tyrosine kinase activation.

Dietary choline effects on milk yield and duodenal choline flow in dairy cattle.

In Experiment 1, the effect, in early lactation, of 0 or 3 g of supplemental choline/kg of total diet DM on milk yield and composition was tested in 20 first lactation and older Holstein cows. In Experiment 2, 30 first lactation and older Holstein cows between 45 and 200 d postpartum were assigned to treatments of 0, 2.5, and 5.0 g of supplemental choline/kg of total diet DM to test the effect of dietary choline with diets based on corn and soybean meal. In Experiments 1 and 2, added choline had no effect on either milk yield or fat-corrected milk yield. In both experiments, fat yield and fat percentage tended to increase with choline supplementation, but protein yield and protein percentage were unaffected. In Experiment 1, choline had no effect on serum lipids. Ruminal dosing of steers with 27 g/d supplemental choline in Experiment 3 increased duodenal choline flow by only 3 g/d. The apparent rumen degradability of choline tended to be higher (77.1 vs. 70.6%) in the supplemented steers. Choline concentration in rumen fluid and duodenal chyme were higher in the supplemented steers. Choline supplementation in Experiments 1 and 3 had no effect on rumen VFA or rumen pH. Dietary choline supplementation apparently is ineffective because of rapid degradation of choline in the rumen.