Regulation of intracellular polyamine biosynthesis and transport by NO and cytokines TNF-alpha and IFN-gamma

Nitric oxide (NO)has been described to exert cytostatic effects on cellularproliferation; however the mechanisms responsible for these effectshave yet to be fully resolved. Polyamines, conversely, are requiredcomponents of cellular proliferation. In experimental models ofinflammation, a relationship between these two pathways has beensuggested by the temporal regulation of a common precursor, arginine.This study was undertaken to determine the effects NO and the NOsynthase (NOS)-inducing cytokines, tumor necrosis factor- (TNF-)and interferon- (IFN-), exert on polyamine regulation. Thetransformed kidney proximal tubule cell line, MCT, maintains highconstitutive levels of the first polyamine biosynthetic enzyme, ornithine decarboxylase (ODC). NO donors markedly suppressed ODC activity in MCT and all other cell lines examined. TNF- and IFN- induction of NO generation resulted in suppressed ODC activity, aneffect prevented by the inducible NOS inhibitorL-N⁶-(1-iminoethyl)lysine(L-NIL). Dithiothreitol reversalof NO-mediated ODC suppression supports nitrosylation as the mechanismof inactivation. We also evaluated polyamine uptake, inasmuch asinhibition of ODC can result in a compensatory induction of polyaminetransporters. Administration of NO donors, or TNF- and IFN-,suppressed[³H]putrescine uptake,thereby preventing transport-mediated reestablishment of intracellularpolyamine levels. This study demonstrates the capacity of NO andinflammatory cytokines to regulate both polyamine biosynthesis and transport.     

Kidney growth,hypertrophy and the unifying mechanism of diabetic complications

Summary. Michael Brownlee has proposed a ‘Unifying Mechanism’ of hyperglycemia-induced damage in diabetes mellitus. At the crux of this hypothesis is the generation of reactive oxygen species (ROS), and their impact on glycolytic pathways. Diabetes is the leading cause of chronic kidney failure. In the early phase of diabetes, prior to establishment of proteinuria or fibrosis, comes kidney growth and hyperfiltration. This early growth phase consists of an early period of hyperplasia followed by hypertrophy. Hypertrophy also contributes to cellular oxidative stress, and may precede the ROS perturbation of glycolytic pathways described in the Brownlee proposal. This increase in growth promotes hyperfiltration, and along with the hypertrophic phenotype appears required for hyperglycemia-induced cell damage and the progression of downstream diabetic complications. Here we will evaluate this growth phenomenon in the context of diabetes mellitus.     

Microcapillary-like structures prompted by phospholipase A2 activation in endothelial cells and pericytes co-cultures on a polyhydroxymethylsiloxane thin film

A thin film of poly(hydroxymethylsiloxane) (PHMS) has been deposited on glass dishes and tested as artificial support material for vascularization from mixed cultures of endothelial cells (EC) and pericytes (PC). The EC/PC co-cultures adhered massively on PHMS, with the formation of net-like microcapillary structures. Such evidence was not found on control glass substrates in the same co-culture conditions neither on PHMS for EC and PC in monocultures. The physicochemical characterization of PHMS and control glass surface by time-of-flight secondary ion mass spectrometry, X-ray photoelectron spectroscopy, water contact angle and atomic force microscopy, pointed to the main role of the polymer hydrophobilicy to explain the observed cellular behavior. Moreover, enhanced intercellular cross-talk was evidenced by the up-regulation and activation of cytoplasmic and Ca(2+)-independent phospholipase A(2) (cPLA(2) and iPLA(2)) expression and cPLA(2) phosphorylation, leading to the cell proliferation and microcapillary formation on the PHMS surface, as evidenced by confocal microscopy analyses. Co-cultures, established with growth-arrested PCs by treatment with mitomycin C, showed an increase in EC proliferation on PHMS. AACOCF(3) or co-transfection with cPLA(2) and iPLA(2)siRNA reduced cell proliferation. The results highlight the major role played by EC/PC cross-talk as well as the hydrophobic character of the substrate surface, to promote microcapillary formation. Our findings suggest an attractive strategy for vascular tissue engineering and provide new details on the interplay of artificial substrates and capillary formation.     

Primary kidney growth and its consequences at the onset of diabetes mellitus

Summary. Diabetes mellitus is a primary contributor to progressive kidney dysfunction leading to end-stage renal disease (ESRD). In the early phase of diabetes, prior to the onset of further complications, both kidney size and glomerular filtration rate (GFR) increase. Glomerular hyperfiltration is considered a risk factor for downstream complications and progression to ESRD. Abnormalities in vascular control have been purported to account for the glomerular hyperfiltration in early diabetes. In this review we discuss a tubulo-centric concept in which tubular growth and subsequent hyper-reabsorption contribute to the onset of glomerular hyperfiltration that demarks the early stage of diabetes. Kidney growth, in this concept, is no longer relegated to a compensatory response to hyperfiltration, but rather plays a primary and active role in its genesis and progression. As such, components of kidney growth, such as the polyamines, may provide a means of early detection of diabetic kidney dysfunction and more effective therapeutic intervention.     

Human serum albumin adsorption onto a-SiC:H and a-C:H thin films deposited by plasma enhanced chemical vapor deposition

In the present paper, we report the study of the adsorption behavior of a model protein such as human serum albumin (HSA) onto surfaces of a-SiC:H and a-C:H thin films deposited by using the plasma-enhanced chemical vapor deposition (PECVD) technique. The surface composition and surface energy of the various substrates as well as the evaluation of the adsorbed amount of protein has been carried out by means of X-ray photoelectron spectroscopy (XPS) and contact angle measurements. It has been found that HSA tends to preferentially adsorb on Si-rich surfaces, as far as the relative amount of adsorbed HSA decreases with increasing S-C concentration. Preliminary elements of mechanistic models are proposed for the correlation between chemical factors and the observed protein adsorption behavior.     

Polyamine transport system mediates agmatine transport in mammalian cells

Agmatine is a biogenic amine with the capacity toregulate a number of nonreceptor-mediated functions in mammalian cells, including intracellular polyamine content and nitric oxide generation. We observed avid incorporation of agmatine into several mammalian celllines and herein characterize agmatine transport in mammalian cells. Intransformed NIH/3T3 cells, agmatine uptake is energy dependent with asaturable component indicative of carrier-mediated transport. Transportdisplays an apparent Michaelis-Menten constant of 2.5 µM and amaximal velocity of 280 pmol · min¹ · mg¹ proteinand requires a membrane potential across the plasma membrane foruptake. Competition with polyamines, but not cationic molecules thatutilize the y⁺ system transporter, suppresses agmatineuptake. Altering polyamine transporter activity results in parallelchanges in polyamine and agmatine uptake. Furthermore, agmatine uptakeis abrogated in a polyamine transport-deficient human carcinoma cellline. These lines of evidence demonstrate that agmatine utilizes, and is dependent on, the polyamine transporter for cellular uptake. Thefact that this transport system is associated with proliferation couldbe of consequence to the antiproliferative effects of agmatine.

     

Distinct domains of CD98hc regulate integrins and amino acid transport

CD98 is a cell surface heterodimer formed by the covalent linkage of CD98 heavy chain (CD98hc) with several different light chains to form amino acid transporters. CD98hc also binds specifically to the integrin beta(1A) cytoplasmic domain and regulates integrin function. In this study, we examined the relationship between the ability of CD98hc to stimulate amino acid transport and to affect integrin function. By constructing chimeras with CD98hc and a type II transmembrane protein (CD69), we found that the cytoplasmic and transmembrane domains of CD98hc are required for its effects on integrin function, while the extracellular domain is required for stimulation of isoleucine transport. Consequently, the capacity to promote amino acid transport is not required for CD98hc's effect on integrin function. Furthermore, a mutant of CD98hc that lacks its integrin binding site can still promote increased isoleucine transport. Thus, these two functions of CD98hc are separable and require distinct domains of the protein.     

Polyphasic changes in incorporation of precursors into ribonucleic acid of oestradiol-stimulated mammary gland

1. At 3 weeks after ovariectomy, mammary glands (5th pair) of adult Swiss mice show (i) no significant decrease in weight, (ii) 20% of the original rate of incorporation of [(3)H]-uridine into RNA (after a 30min pulse), and (iii) 90% of the original rate of incorporation of l-[(3)H]leucine into protein (after a 15min pulse). 2. A single injection of oestradiol-17beta into these ovariectomized mice produces, during the next 17h, a series of discrete bursts of increased incorporation of [(3)H]uridine into mammary-gland RNA; the bursts, which are variable in height, reach peaks at approx. 1, 9, 12 and 16h after hormone administration; an increase is already detected at 15min, the earliest time-point investigated; each burst lasts for approx. 2h. There is no significant stimulation of [(3)H]uridine incorporation into RNA of liver and quadriceps femoris muscle. 3. Nuclear incorporation of [(3)H]UTP into RNA of mammary gland in vitro is linear with time for up to 20min at 15 degrees C; it requires CTP, GTP and ATP and is inhibited by actinomycin D. Also, the incorporation is strongly inhibited by alpha-amanitin in high salt concentrations but only weakly in low salt concentrations, a result indicating that RNA polymerase II activity predominates in high salt, whereas RNA polymerase I activity predominates in low salt concentrations. Injection of oestradiol-17beta in vivo followed by measurement of nuclear RNA synthesis in vitro shows a definite increase in both RNA polymerase activities 30min after oestradiol-17beta injection, the earliest time-point investigated, a higher increase at 1h, a decline at 4h, and again a large increase at 12h. These results in general agree with the changes in precursor incorporation into RNA measured directly in the animal and suggest that changes in [(3)H]uridine uptake into RNA are not precursor-pool-dependent.     

Agmatine prevents the Ca2+-dependent induction of permeability transition in rat brain mitochondria

The arginine metabolite agmatine is able to protect brain mitochondria against the drop in energy capacity by the Ca²⁺-dependent induction of permeability transition (MPT) in rat brain mitochondria. At normal levels, the amine maintains the respiratory control index and ADP/O ratio and prevents mitochondrial colloid-osmotic swelling and any electrical potential (ΔΨ) drop. MPT is due to oxidative stress induced by the interaction of Ca²⁺ with the mitochondrial membrane, leading to the production of hydrogen peroxide and, subsequently, other reactive oxygen species (ROS) such as hydroxyl radicals. This production of ROS induces oxidation of sulfhydryl groups, in particular those of two critical cysteines, most probably located on adenine nucleotide translocase, and also oxidation of pyridine nucleotides, resulting in transition pore opening. The protective effect of agmatine is attributable to a scavenging effect on the most toxic ROS, i.e., the hydroxyl radical, thus preventing oxidative stress and consequent bioenergetic collapse.