Allan L. Campione

Glycine transport in mouse eggs ad preimplantation conceptuses

At least two Na+-dependent systems for glycine transport became detectable, while another became undetectable during preimplantation development of mouse conceptuses. Glycine was taken up by a process in eggs and cleavage-stage conceptuses which closely resembles system Gly. Mediated transport at these stages was more rapid at higher Cl- concentrations, sigmoidally related to the exogenous Na+ concentration, and strongly inhibited by sarcosine but not by amino acids with larger side chains. Moreover, neither Li+ nor choline could substitute for Na+ in stimulating glycine transport. System Gly was the only mediated process detected for glycine uptake in unfertilized and fertilized eggs and two-cell conceptuses, but two, less conspicuous, sarcosine-resistant, Na+-dependent components of transport also appeared to be present in eight-cell conceptuses. One of the latter components seemed to remain relatively inconspicuous when conceptuses formed blastocysts, while system Gly became undetectable. In contrast, the other less conspicuous component in eight-cell conceptuses appeared to become the most conspicuous transport process in blastocysts. The latter process, previously designated system B0,+, was shown here also to interact strongly with a broad scope of zwitterionic and cationic amino acid structures. Moreover, transport of glycine via system B0,+ was more rapid at higher Cl- concentrations, and this Na+-dependent process as well as Na+-independent leucine uptake were inhibited by choline. Furthermore, Na+-dependent amino acid transport in two-cell conceptuses and blastocysts was inhibited by 1.0 or 10 mM ouabain, but the inhibition was incomplete at both concentrations. Since Na+/K+-ATPase has not been detected in two-cell conceptuses, inhibition of amino acid transport by ouabain may not have been due solely to an effect on this enzyme. The level of system Gly activity decreased during the development of eight-cell conceptuses from eggs, and this decrease could contribute to an associated decline in intracellular glycine. Since other amino acids begin to compete strongly with glycine for transport when system B0,+ replaces system Gly in conceptuses, this qualitative change in transport activity may help account for a further decrease in the glycine content of conceptuses, reported elsewhere to occur after they form blastocysts.

Ouabain-sensitive Rb+ uptake in mouse eggs and preimplantation conceptuses.

The results of histochemical and immunocytochemical studies have been used elsewhere to support the hypothesis that Na+/K(+)-ATPase expression is initiated or increases dramatically in preimplantation mouse conceptuses just before they begin to cavitate. Moreover, localization of the enzyme in the inner membrane of the mural trophoblast is thought to be involved directly in formation and maintenance of the blastocyst cavity. Presumably, Na+/K(+)-ATPase extrudes the cation, Na+, and therefore water into the cavity. The cation transporting activity of the enzyme can be determined by measuring ouabain-sensitive Rb+ uptake by cells. Therefore, we measured Rb+ uptake in mouse eggs and preimplantation conceptuses at various stages of development. 86Rb+ uptake by conceptuses increased linearly with time for at least 60 min in medium containing 0.7 mM total Rb+ plus K+ in the absence or presence of 1.0 mM ouabain, and ouabain inhibited more than 70% of 86Rb+ uptake. The ouabain concentration at 1/2 of maximum inhibition of the ouabain-sensitive component of 86Rb+ uptake was about 10-20 microM in eggs and conceptuses at all stages of preimplantation development. Moreover, ouabain-sensitive Rb+ uptake had a twofold higher Vmax value in blastocysts than in eggs or conceptuses at earlier stages of development (i.e., approximately 173 vs 70-100 fmole.conceptus-1.min-1), although the total cell surface area also was probably about two times greater in blastocysts than in eggs or other conceptuses. Ouabain-sensitive Rb+ transport in eggs and conceptuses may have occurred via a single ouabain-sensitive Rb+ transporter with a Hill coefficient of 1.5-1.8 (Hill plots). When it was assumed that the Hill coefficient had a value of 2.0, however, eggs and conceptuses appeared to contain at least two forms of Na+/K(+)-ATPase activity. These studies are the first to show that the cation transporting activity of Na+/K(+)-ATPase can be measured quantitatively in mammalian eggs and preimplantation conceptuses. Inclusion of this assay in experiments designed to determine how Na+/K(+)-ATPase activity is controlled in oocytes and conceptuses should yield further insight into the role of this enzyme in oogenesis and preimplantation development.

Changes in the activities of amino acid transport systems b0,+ and L during development of preimplantation mouse conceptuses.

Uptake of leucine, lysine, and arginine was predominantly Na(+)-independent in mouse conceptuses through the 8-cell stage of development, and two components of saturable transport were detected for each of these amino acids. Uptake of cationic substrates from solutions near 1 microM was inhibited most strongly by bulky cationic and zwitterionic amino acids whose carbon skeletons do not branch at the alpha or beta positions. By this criterion, system b0,+ accounted for most of the Na(+)-independent arginine and lysine transport in eggs and conceptuses throughout preimplantation development. A small, leucine-resistant, cation-preferring component of amino acid transport was also detected in these cells. Leucine uptake was inhibited most strongly by bicyclic, branched-chain or benzenoid, zwitterionic amino acids in eggs and conceptuses prior to formation of blastocysts. Therefore, it appeared to be taken up mainly by system L, while system b0,+ accounted for a smaller portion of leucine uptake during this developmental period. In blastocysts, in contrast, system L was less conspicuous, and system b0,+ was primarily responsible for Na(+)-independent leucine uptake. The Vmax values for transport of amino acids by system b0,+ increased by up to 30-fold in conceptuses between the 1-cell and blastocyst stages. In contrast, the Vmax value for leucine transport via system L decreased while the Km value increased between these two developmental stages. Although several explanations for these changes are possible, we favor the hypothesis that the density of system L transport sites in plasma membranes decreases while the number of system b0,+ sites increases during development of blastocysts from 1-cell conceptuses.

Functional changes in cation-preferring amino acid transport during development of preimplantation mouse conceptuses.

In a previous study, a Na(+)-independent, cation-preferring amino acid transport system was detected in preimplantation mouse blastocysts. The system resisted Na(+)-dependent inhibition by homoserine and so resembled the lysosomal system c more than it resembled the plasmalemmal system y+. We now report the presence of a cation-preferring system in unfertilized and fertilized eggs and cleavage-state conceptuses which also resists Na(+)-dependent inhibition by homoserine. The systems in 1-cell conceptuses and blastocysts are, however, insensitive to changes in pH in the interval of 6.0 to 8.0 and, thus, different from the pH-sensitive system c. Moreover, the relative strengths of the interactions of a variety of basic amino acids with the systems in conceptuses do not correspond well with the relative strengths of their interactions with either system c or system y+. Similarly, the system in 1-cell conceptuses can be distinguished from the system in blastocysts because L-arginine interacts about equally well with each of these systems, whereas the system in 1-cell conceptuses is inhibited more strongly than the system in blastocysts by most other basic amino acids. In addition, inhibition of the system in 1-cell conceptuses by some basic amino acids is Na(+)-stimulated, whereas Na+ does not affect inhibition of the system in blastocysts. Finally, L-tryptophan inhibits the system in blastocysts better than L-histidine or D-arginine do, but the reverse is true for the system in 1-cell conceptuses. Therefore, the relative activities of at least two forms of a novel, cation preferring amino acid transport process change during development of blastocysts from fertilized eggs. For convenience, the forms of the cation-preferring transport processes that seem to predominate at the 1-cell and blastocysts stages are provisionally designated systems b+1 and b+2, respectively, although these two systems need not represent entirely different gene products.

Vitamin C protects low-density lipoprotein from homocysteine-mediated oxidation.

Homocysteine, an atherogenic amino acid, promotes iron-dependent oxidation of low-density lipoprotein (LDL). We investigated whether vitamin C, a physiological antioxidant, could protect LDL from homocysteine-mediated oxidation. LDL (0.2 mg of protein/ml) was incubated at 37 degrees C with homocysteine (1000 microM) and ferric iron (10-100 microM) in either the absence (control) or presence of vitamin C (5-250 microM). Under these conditions, vitamin C protected LDL from oxidation as evidenced by an increased lag time preceding lipid diene formation (> or = 5 vs. 2.5 h for control), decreased thiobarbituric acid-reactive substances accumulation (< or = 19 +/- 1 nmol/mg when vitamin C > or = 10 microM vs. 32 +/- 3 nmol/mg for control, p <.01), and decreased lipoprotein anodic electrophoretic mobility. Near-maximal protection was observed at vitamin C concentrations similar to those in human blood (50-100 microM); also, some protection was observed even at low concentrations (5-10 microM). This effect resulted neither from altered iron redox chemistry nor enhanced recycling of vitamin E in LDL. Instead, similar to previous reports for copper-dependent LDL oxidation, we found that vitamin C protected LDL from homocysteine-mediated oxidation through covalent lipoprotein modification involving dehydroascorbic acid. Protection of LDL from homocysteine-mediated oxidation by vitamin C may have implications for the prevention of cardiovascular disease.

Development of amino acid transport system B0,+ in mouse blastocysts

The capacity of preimplantation mouse blastocysts to express the novel amino acid transport activity provisionally designated system B0,+ increased approximately 3-fold 1 day after administration of estrogen to their progesterone-primed, ovariectomized mothers. Nevertheless, blastocysts obtained 22-25 h after estrogen administration (implanting blastocysts) had to be incubated in vitro for about 20 min before they fully expressed their B0,+ activity. No similar increase in B0,+ activity was observed upon incubation of blastocysts obtained before estrogen administration (diapausing blastocysts). Rapid metabolic changes can be induced in the uterus by massaging it with a blunt instrument while it is receptive to implantation, and this treatment was found to increase the apparent B0,+ activity in implanting but not diapausing blastocysts. In contrast, the activity of an incompletely characterized, Na+-independent system, which accepts L-lysine as a substrate, decreased more than 2-fold when implanting blastocysts were incubated in vitro. No change in Na+-independent lysine uptake was detected during incubation of diapausing blastocysts. It is suggested that both uteri and blastocysts develop the capacity to change rapidly some of their metabolic processes near the time of implantation, and one of the processes which may be subject to rapid change in blastocysts is amino acid transport. These developmental events appear to coincide with and could be required for the decidual cell response and implantation of blastocysts in the uterus.

Plasma thiols inhibit hemin-dependent oxidation of human low-density lipoprotein.

Oxidative modification of human low-density lipoprotein (LDL) renders it atherogenic. Previous studies demonstrated that plasma thiols promote oxidation of LDL by free ferric iron (Fe3+). The current study investigated effects of plasma thiols on oxidation of LDL by hemin, a physiological Fe3+-protoporphyrin IX complex thought to be capable of initiating LDL oxidation in vivo. In contrast to free Fe3+ which is incapable of oxidizing LDL in the absence of an exogenous reductant, hemin readily promoted LDL oxidation. During incubation of LDL (0.2 mg of protein/ml) with hemin (10 microM) at 37 degrees C for 6 h, thiobarbituric acid-reactive substances (TBARS), a marker of lipid oxidation, increased from 0.3 (+/-0.1) nmol/mg of LDL protein to a maximal concentration of 45.8 (+/-5.2) nmol/mg of LDL protein. Under the same experimental conditions, lipid-conjugated dienes, another marker of lipid oxidation, increased from non-detectable to near-maximal levels of 78-187 nmol/mg of LDL protein, and lipoprotein polyunsaturated fatty acyl-containing cholesteryl ester content decreased to 15-36% of that present in native (i.e. unoxidized) LDL. Continued incubation of LDL with hemin for up to 24 h resulted in no further significant alterations in lipoprotein levels of TBARS, lipid-conjugated dienes, and cholesteryl esters. In addition to these chemical modifications indicative of lipoprotein oxidation, agarose gel electrophoretic analysis indicated that exposure of LDL to hemin resulted in conversion of the lipoprotein to an atherogenic form as evidenced by its increased anodic electrophoretic mobility. Addition of physiological concentrations of plasma thiols (either cysteine, homocysteine or reduced glutathione; 1-100 microM, each) inhibited hemin-mediated oxidation of LDL. Thus, whereas the maximal TBARS concentration was achieved following 6 h of incubation of LDL with hemin alone, addition of thiol extended the time required to attain maximal TBARS concentration to > or = 12 h. Similar antioxidant effects of thiols on formation of lipid-conjugated dienes, loss of cholesteryl esters, and lipoprotein anodic electrophoretic mobility were also observed. However, all thiols were not equally effective at inhibiting hemin-dependent LDL oxidation. Thus, whereas reduced glutathione was most effective at inhibiting hemin-dependent LDL oxidation, an intermediate effect was observed for homocysteine, and cysteine was least effective. The inhibition of hemin-mediated LDL oxidation by plasma thiols reported here confirms a previous observation that, under certain conditions, thiols can function as antioxidants, but contrasts with the previously documented pro-oxidant effect of the same thiols on oxidation of LDL by free Fe3+. These contrasting effects of plasma thiols on hemin- and free Fe3+-mediated LDL oxidation indicate that, in vivo, the ability of thiols to function as either anti- or pro-oxidants during LDL oxidation may, at least in part, be determined by the type of oxidant stress to which the lipoprotein is exposed.

AMINO ACID TRANSPORT REGULATION IN PREIMPLANTATION MOUSE EMBRYOS: EFFECTS ON AMINO ACID CONTENT AND PRE- AND PERI-IMPLANTATION DEVELOPMENT

Several nonessential amino acids improve preimplantation development of mouse embryos in vitro and increase the proportion of the embryos that implant after transfer to surrogate mothers. The beneficial effects of the amino acids are associated with accumulation of them by embryos via transport systems that select for the amino acids. Expression of most of the transport systems is developmentally regulated apparently at the level of synthesis and degradation of mRNAs encoding their transport proteins. Since genes encoding pertinent amino acid transport proteins have now been partially cloned, it will be possible to use transgenic experiments further to test the theory that regulation of amino acid transport is needed for normal pre- and peri-implantation development.

p53 transactivity during in vitro osteoblast differentiation in a rat osteosarcoma cell line.

We previously demonstrated a correlation between wild‐type p53 expression and appearance of osteoblastic‐specific differentiation characteristics, as evidenced by basal osteocalcin gene expression in a mouse osteosarcoma tumor. The study reported here further explored the possibility of p53s having a distinct transcription‐activating role in bone differentiation, in addition to its proposed role in G1 arrest and apoptosis. ROS17/2.3 osteoblastic osteosarcoma cells were stably transfected with a plasmid containing wild‐type p53 binding sequences fused to the chloramphenicol acetyltransferase reporter gene. These cells were used to determine the transactivating role of p53 in regulation of osteocalcin gene expression. We chose two conditions under which osteocalcin expression is known to be upregulated: exposure of osteoblastic cells to differentiation‐promoting medium and to vitamin D3. Exposure of the transfected cells to differentiation‐promoting medium produced an increase in p53 transactivating activity correlating with the appearance of osteocalcin expression after about 1 wk. Vitamin D3 treatment resulted in upregulation of osteocalcin activity without a corresponding change in p53 transactivation activity or expression. In separate experiments, we tested whether changes in osteocalcin expression accompanied changes in p53 activity under conditions of downregulation of cell proliferation mediated by inhibition of DNA synthesis. Hydroxyurea treatment was used to inhibit DNA synthesis and produce growth arrest in osteoblastic cells. Inhibition of osteoblast cell proliferation was associated with a fourfold increase in p53 transactivating activity and a transient increase in osteocalcin steady‐state expression. These results demonstrated a close relationship between p53 and osteocalcin and suggested a regulatory role for wild‐type p53 in the control of basal osteocalcin gene expression in osteoblasts. Mol. Carcinog. 25:132–138, 1999.

Transport of benzenoid amino acids by system T and four broad scope systems in preimplantation mouse conceptuses

We have studied transport of L-tryptophan, L-tyrosine and L-phenylalanine as factors contributing to homeostasis of these amino acids in preimplantation mouse conceptuses. Benzenoid amino acids were transported by the Na(+)-independent systems L and b0,+ in 1-cell conceptuses, and by these systems plus the Na(+)-dependent systems B0,+ and B in blastocysts. In addition, a component of Na(+)-independent tryptophan, tyrosine and phenylalanine transport in 1-cell and 2-cell conceptuses and in blastocysts resisted inhibition by L-leucine. The latter component of transport not only preferred benzenoid amino acids and in particular tryptophan as substrates, but it also was inhibited strongly and competitively by alpha-N-methyl-L-tryptophan. The leucine-resistant component of tryptophan transport also was inhibited strongly by N-ethylmaleimide and D-tryptophan, and it appeared to be inhibited weakly by 3-amino-endo-bicyclo[3.2.1]octane-3-carboxylic acid (BCO) but not by other amino acids tested as inhibitors. By these criteria, the leucine-resistant component of transport of benzenoid amino acids resembled system T in human red blood cells and rat hepatocytes. It is not entirely clear why preimplantation blastocysts have five good systems for transport of tryptophan. It is possible, however, that tryptophan homeostasis is particularly important during preimplantation development since it has been shown elsewhere that tryptophan availability in blood increases within one day after rat eggs are fertilized.