Prevention of chronic anthracycline cardiotoxicity in the adult Fischer 344 rat by dexrazoxane and effects on iron metabolism.

PURPOSE: Anthracyclines, such as doxorubicin and daunorubicin, continue to be widely used in the treatment of cancer, although they share the adverse effect of chronic, cumulative dose-related cardiotoxicity. The only approved treatment in prevention of anthracycline cardiotoxicity is dexrazoxane, a putative iron chelator. Previous in vitro studies have shown that disorders of iron metabolism, including altered IRP1-IRE binding, may be an important mechanism of anthracycline cardiotoxicity. METHODS: This study examined the role of IRP1-IRE binding ex vivo in a chronic model of daunorubicin cardiotoxicity in the Fischer 344 rat and whether dexrazoxane could prevent any daunorubicin-induced changes in IRP1 binding. Young adult (5-6 months) Fischer 344 rats received daunorubicin (2.5 mg/kg iv once per week for 6 weeks) with and without pretreatment with dexrazoxane (50 mg/kg ip). Other groups received saline (controls) or dexrazoxane alone. Rats were killed either 4 h or 2 weeks after the last dose of daunorubicin to assess IRP1-IRE binding. RESULTS: Contractility (dF/dt) of atrial tissue, obtained from rats 2 weeks after the last dose of daunorubicin, was significantly reduced in daunorubicin-treated compared to control rats. Dexrazoxane pretreatment protected against the daunorubicin-induced decrease in atrial dF/dt. However, left ventricular IRP1/IRE binding was not affected by daunorubicin treatment either 4 h or 2 weeks after the last dose of daunorubicin. CONCLUSIONS: IRP1 binding may not be altered in the rat model of chronic anthracycline cardiotoxicity.

CD40 is functionally expressed on human breast carcinomas: variable inducibility by cytokines and enhancement of Fas-mediated apoptosis.

The CD40 molecule, a member of the TNF receptor gene family, has been intensively studied with respect to regulation of B cell proliferation and survival. Although CD40 is also expressed on carcinoma cell lines, information concerning the biological function of CD40 on cells of epithelial origin is limited. In this study we detected constitutive CD40 on human breast carcinoma cell lines and an increase in CD40 expression following treatment with cytokines IL-1alpha and IFN-gamma. CD40 ligation was also found to increase MHC II expression in cells pretreated with IFN-gamma. In contrast to normal B cells, where CD40 signaling provides a potent survival signal, we observed that CD40 ligation in breast carcinoma cells results in growth inhibition and enhanced susceptibility to Fas-mediated apoptosis. Enhanced apoptosis appears to be attributable, at least in part, to an up-regulation of Fas expression caused by CD40 ligation. These results suggest a potentially important role for CD40 in breast tumor biology.

Oncostatin M-specific receptor expression and function in regulating cell proliferation of normal and malignant mammary epithelial cells.

Oncostatin M (OSM) is a cytokine produced by activated T lymphocytes and macrophages. OSM is structurally and functionally related to leukaemia inhibitory factor (LIF), another cytokine in the interleukin 6 (IL-6) family. The biological activities of OSM are mediated through two types of receptor complexes, the LIF/OSM shared receptor (type I) and OSM-specific receptor (OSM-R, type II), which is composed of gp130 as a binding subunit and a newly identified affinity conversion subunit, OSM-R beta. Previous research conducted in the authors' laboratory has shown that OSM inhibits the growth of several breast cancer cell lines. To investigate whether OSM has a similar effect in primary normal human mammary epithelial (HME) cells, the activity of OSM in HME cells derived from four donors was examined. OSM produced a dose-dependent inhibition of DNA synethesis in these cells. In order to determine the receptor subtypes mediating OSM activity in HME and breast cancer cells, flow cytometry analysis using anti-gp130mAb and anti-OSM-R beta mAb was performed. In these studies, the authors were able to examine expressions of gp130 and OSM-R beta. In addition, quantitative RT-PCR assays were conducted to measure expressions of the mRNAs of the subunits for type I and type II OSM receptor. The results show that HME cells and most breast cancer cell lines express both the type I and the type II OSM receptors. However, type II, OSM-specific receptors are expressed at a higher levels than type I, OSM/LIF shared receptors. Accordingly, we compared the growth regulatory activities of OSM with LIF in HME cells and in breast cancer cells. In contrast to the inhibitory activity of OSM, LIF stimulated the growth of breast cancer cells, whereas it had no effect on normal mammary epithelial cell growth. Together, these data suggest that OSM plays an inhibitory role in normal and malignant mammary epithelial cell growth in vitro. OSM activity is mediated by the OSM-specific receptor (type II), not by the OSM/LIF shared receptor.

Beta-adrenoceptor agonists block corticosteroid inhibition in eosinophils.

Although beta-adrenoceptor agonists are primary agents in therapy of asthma, epidemiological studies have suggested that frequent or prolonged used of these drugs could be associated with exacerbation of disease. Mechanisms of any adverse effects remain unclear although in vitro studies have suggested that beta-adrenoceptor agonists can block glucocorticoid actions. Because asthma is an inflammatory disease characterized by eosinophil infiltration of the airways, actions of beta-agonists and corticosteroids that alter eosinophil survival and mediator generation may be of importance. Eosinophil generation of superoxide anion, a potent mediator that can damage respiratory epithelium, was markedly increased after 2-24 h of in vitro beta-adrenoceptor agonist exposure. These proinflammatory effects are in contrast to inhibition of superoxide generation, which is observed with acute beta-agonist exposure. Corticosteroid treatment to reduce inflammation is combined with beta-agonist therapy in current asthma guidelines. Although dexamethasone independently decreased eosinophil superoxide anion generation, in the presence of beta-adrenoceptor agonist dexamethasone inhibition was minimal and not statistically significant. Eosinophil survival is a relevant factor to pulmonary inflammation. Although beta-adrenoceptor agonists did not independently increase eosinophil survival, glucocorticoid actions that increase apoptosis were blocked. Thus, in vitro beta-agonists can independently increase inflammatory mediator generation and block anti-inflammatory actions of corticosteroid.

Beta-adrenergic regulation of the eosinophil respiratory burst as detected by lucigenin-dependent luminescence.

Because beta-adrenoceptor agonists are commonly used in the treatment of disease states associated with eosinophil activation, beta-adrenergic regulation of the eosinophil respiratory burst (as monitored with lucigenin-dependent luminescence) was evaluated. Normodense, nonprimed eosinophils from healthy volunteer subjects were potently inhibited by very low concentrations of isoproterenol. The inhibitory concentration of 50% for isoproterenol was approximately 2 nmol/L. The beta-agonist was able to inhibit the eosinophil respiratory burst induced by receptor-mediated (chemotactic peptide) and nonreceptor-mediated (calcium ionophore and phorbol ester) stimuli. Thus beta-agonist inhibition was unlikely to be isolated to an effect at the receptor or G protein linkage. To determine whether cyclic adenosine 3',5' monophosphate (cAMP) may mediate beta-agonist effects, studies were performed with the type IV cAMP phosphodiesterase inhibitor Ro-201724. beta-Agonist inhibition of the respiratory burst was clearly synergistic with effects of Ro-201724. We conclude that beta-adrenoceptor agonists can regulate the eosinophil respiratory burst at least partially through an effect mediated by cAMP. Because regulation of the eosinophil by isoproterenol was observed at very low concentrations, these results may be relevant to pharmacologic effects of beta-agonists in disease states complicated by eosinophil activation during asthma.

Regulation of the respiratory burst by cyclic 3',5'-AMP, an association with inhibition of arachidonic acid release.

The mechanism of cAMP regulation of the respiratory burst was studied with HL-60 cells that had been DMSO-differentiated to a neutrophil-like cell. To evaluate the effects of known cAMP concentrations, cells were permeabilized with streptolysin-O. Chemotactic peptide (FMLP)-stimulated NADPH oxidase activity was inhibited by cAMP at concentrations higher than 3 microM. Because intracellular calcium was buffered, inhibitory actions of cAMP were not mediated by modulation of calcium concentration. Effects of cAMP on chemotactic peptide signal transduction mediated by phospholipase C, phospholipase D, and phospholipase A2 were then determined. Neither inositol phosphate generation (phospholipase C) nor phosphatidylethanol generation (phospholipase D activity in presence of 1.6% ethanol) induced by FMLP were significantly affected by cAMP. In contrast, cAMP potently inhibited FMLP-induced arachidonic acid mobilization (phospholipase A2). NADPH oxidase activity induced by exogenous arachidonic acid was not inhibited by cAMP. These results indicate that cAMP-mediated inhibition of arachidonic acid mobilization may be important in regulation of the respiratory burst.

Impaired stimulus-response coupling in association with increased growth rate of HL60 cells.

Impairments in the respiratory burst and stimulus-response coupling were studied with respect to the increased rate of cell replication that occurred in HL60 cells during repetitive passages in cell culture. During a 45-week period of culture, HL60 cells developed a progressive increase in rate of replication. Concomitantly, undifferentiated cells developed an impairment in ATP-induced calcium mobilization. The percentage of cells that could be differentiated with dimethyl sulfoxide progressively diminished. Differentiated cells developed an impairment in both the respiratory burst and secretion of beta-glucuronidase. In addition, regulation of the respiratory burst by cAMP agonists including isoproterenol, adenosine, and prostaglandin E2 was reduced in rapidly proliferating cells. Thus, multiple changes in stimulus-response coupling occur during cell culture in association with an increase in rate of cell replication. It may be important to recognize progressive impairments in cell function in studies using repetitive samples of HL60 cells from a continuously maintained cell population. The observed impairments in stimulus-response coupling may be relevant to unregulated cell growth in neoplastic disease.

Inhibition of aldosterone production and angiotensin action by drugs affecting potassium channels.

We screened potassium channel agonists and antagonists in a search for pharmacologic probes of the channels that mediate potassium's effects on adrenal zona glomerulosa cells. Suspensions of bovine cells were tested, and aldosterone was measured by radioimmunoassay. The most potent inhibitors were pinacidil, capsaicin, glyburide, and quinine. These reagents were more potent against aldosterone production than against cortisol production. Aldosterone produced under basal conditions, as well as that stimulated by potassium, angiotensin II, or dibutyryl cyclic AMP, was antagonized. The vasodilatory and aldosterone-inhibiting potencies of potassium channel reagents were very different. Candidate antihypertensives with potassium channel activity should be tested for adrenal inhibition.

Ionophores for monovalent cations inhibit angiotensin-stimulated aldosteronogenesis.

We examined the effects of monensin (a sodium ionophore), valinomycin (a potassium ionophore), and nigericin (a nonspecific ionophore) on steroid production and its stimulation in bovine adrenal glomerulosa and fasciculata cells. All three ionophores at nanomolar concentrations inhibited angiotensin (AII)-stimulated aldosterone production; potassium-stimulated aldosteronogenesis was more sensitive, and cortisol synthesis by fasciculata cells was much less sensitive. Ionophores completely inhibited the early pathway of aldosteronogenesis and partially inhibited conversion of progesterone to aldosterone. Ionophores had no effect on pregnenolone production by isolated glomerulosa mitochondria. Monensin had no effect on AII binding, calcium flux, calcium transient, protein phosphorylation, or protein synthesis; valinomycin slightly inhibited these processes. Valinomycin lowered cell potassium and raised cell sodium, but its inhibition of aldosteronogenesis was not overcome by increasing extracellular potassium. Monensin and nigericin had no effect on cell potassium or sodium. Cellular ATP was decreased by valinomycin, but not by monensin or nigericin. Our results show that stimulation of aldosteronogenesis by AII and potassium is highly sensitive to ionophores of monovalent cations. Monensin and nigericin inhibit steroidogenesis at concentrations that have no other observed deleterious effects on glomeulosa cells. These results identify a distinguishing characteristic of adrenal glomerulosa cells and suggest a new pharmacologic approach to inhibition of aldosteronogenesis.

Effects of ouabain and potassium on protein synthesis and angiotensin-stimulated aldosterone synthesis in bovine adrenal glomerulosa cells.

Ouabain (1 microM and below) inhibited both basal and angiotensin II-stimulated aldosterone synthesis in bovine adrenal glomerulosa cells. Ouabain had no effect on binding of 125I-labeled angiotensin, on angiotensin's effects on 45Ca2+ fluxes, or on 32PO4 incorporation into phosphatidylinositol. This spectrum of activities resembles that of the protein synthesis inhibitor cycloheximide, which also blocks aldosterone synthesis. Ouabain was, therefore, tested for its effect on protein synthesis, as measured by uptake of [3H]leucine into acid-precipitable material. Ouabain inhibited protein synthesis at concentrations similar to those that depressed aldosterone synthesis, but did not block uptake of the nonmetabolized amino acid [carboxyl-14C]aminocyclopentane-1-carboxylic acid, nor the entrance of [3H]leucine into cells. When cells previously loaded with 86Rb+ were treated with 1 microM ouabain, they lost approximately half of the accumulated radioactivity in 30 min. When cells were incubated in potassium-free buffer, both protein and aldosterone synthesis were severely inhibited. Increased extracellular potassium reversed ouabain's inhibition of protein and aldosterone synthesis in parallel. Pregnenolone synthesis was inhibited by ouabain, and elevated potassium overcame that blockade. Ouabain did not block aldosterone synthesis from exogenous progesterone. These data fit a model in which ouabain causes loss of cell potassium, which, in turn, depresses protein synthesis. Since protein synthesis is necessary for angiotensin II stimulation of cholesterol side-chain cleavage, ouabain depresses that step, pregnenolone synthesis, and thus, aldosterone synthesis.

Veratridine, angiotensin receptors and aldosteronogenesis in bovine adrenal glomerulosa cells.

Veratridine inhibited angiotensin binding to its receptors in bovine adrenal glomerulosa cells and vascular smooth muscle. Fifty percent inhibition of adrenal receptors required about 2 X 10(-5) M veratridine. Receptors from vascular tissue were less sensitive. Graphic analysis showed that inhibition resulted primarily from a reduction of receptor number. Angiotensin stimulation of phosphatidylinositol turnover and of aldosterone production was inhibited by veratridine. Analogues of veratridine varied in their receptor inhibitory potency, but these variations did not correlate with the potencies of analogues as hypotensive agents or inhibitors of aldosteronogenesis. Very low extracellular sodium concentrations inhibited the adrenal effects of angiotensin. Neither veratridine nor grayanotoxin, both of which open sodium channels in excitable tissues, had a detectable effect on sodium fluxes in adrenal cells. Inhibition of aldosteronogenesis by veratridine is more likely the result of receptor and post-receptor effects than an alteration of the sodium channel.

Angiotensin effects on calcium and steroidogenesis in adrenal glomerulosa cells.

We investigated the role of cellular calcium pools in angiotensin II-stimulated aldosterone synthesis in bovine adrenal glomerulosa cells. Angiotensin II decreased the size of the exchangeable cell calcium pool by 34%, consistent with previous observations that angiotensin II causes decreased uptake of 45Ca+2 into cells and increased efflux of 45Ca+2 from preloaded cells. Atomic absorption spectroscopy showed that angiotension II caused a decrease of 21% in total cellular calcium. Angiotensin II caused efflux of 45Ca+2 in the presence of EGTA and retarded uptake of 45Ca+2 when choline was substituted for sodium, suggesting that hormone effects on calcium pools do not involve influx of trigger calcium or sodium. Cells incubated in calcium-free buffer and 0.1 mM or 0.5 mM EGTA synthesized reduced (but still significant) amounts of the steroid in response to hormone. Cells incubated in increasing concentrations of extracellular calcium contained increasing amounts of intracellular calcium and synthesized increasing amounts of aldosterone in response to angiotensin II. These results point to the participation of intracellular calcium pools in angiotensin II-stimulated steroidogenesis and the importance of extracellular calcium in maintaining these pools.

Peptidoglycan in the cell wall of the primary intracellular symbiote of the pea aphid.

Primary intracellular symbiotes of the pea aphid, Acyrthosiphon pisum (Harris), when fixed with potassium permanganate, revealed a distinctly staining area between the cytoplasmic membrane and the outer cell-wall envelope. This area is thought to be analogous to the peptidoglycan complex of the Eubacteriales. In addition, the diagnostic bacterial peptidoglycan amino compounds, muramic acid and diaminopimelic acid, were detected in a hydrochloric acid hydrolyzate of isolated symbiotes.