Homocysteine is transported by the microvillous plasma membrane of human placenta.

Elevated maternal plasma concentrations of homocysteine (Hcy) are associated with pregnancy complications and adverse neonatal outcomes. The postulate that we wish to advance here is that placental transport of Hcy, by competing with endogenous amino acids for transporter activity, may account for some of the damaging impacts of Hcy on placental metabolism and function as well as fetal development. In this article, we provide an overview of some recent studies characterising the transport mechanisms for Hcy across the microvillous plasma membrane (MVM) of the syncytiotrophoblast, the transporting epithelium of human placenta. Three Hcy transport systems have been identified, systems L, A and y(+)L. This was accomplished using a strategy of competitive inhibition to investigate the effects of Hcy on the uptake of well-characterised radiolabelled substrates for each transport system into isolated MVM vesicles. The reverse experiments were also performed, examining the effects of model substrates on [³5S]L-Hcy uptake. This article describes the evidence for systems L, A and y(+)L involvement in placental Hcy transport and discusses the physiological implications of these findings with respect to placental function and fetal development.

Homocysteine transport by systems L, A and y+L across the microvillous plasma membrane of human placenta.

Elevated maternal plasma levels of homocysteine (Hcy) are associated with pregnancy complications and adverse neonatal outcomes, suggesting placental transport of Hcy may impact on fetal development. However, such transport mechanisms have not been defined. In this study we characterise Hcy transport mechanisms across the microvillous plasma membrane (MVM) of the syncytiotrophoblast, the transporting epithelium of human placenta. Three candidate transport systems, systems L, A and y(+)L, were examined utilising competitive inhibition to investigate the effects of Hcy on the uptake of well-characterised radiolabelled substrates for each system into isolated MVM vesicles, and that of model substrates on 10 microm [(35)S]l-Hcy uptake. System L activity was inhibited by both l-Hcy and dl-Hcy, comparable to model substrates including 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid (BCH). System L constituted the major transport mechanism, with significant BCH inhibition (69%) of [(35)S]l-Hcy uptake. System A activity was also inhibited by l-Hcy and dl-Hcy with a smaller contribution (21%) to [(35)S]l-Hcy uptake. Inhibition by l-Hcy and dl-Hcy of system y(+)L activity was Na(+) sensitive with a significant inhibition constant (K(i)) shift observed following K(+) replacement; l-arginine reduced [(35)S]l-Hcy uptake by 19%. Kinetic modelling of [(35)S]l-Hcy uptake resolved two, Na(+)-independent, transport components (K(m) 72 microm and 9.7 mm). This study provides evidence for the involvement of systems L, A and y(+)L in placental Hcy transport. Such transport, by competing with endogenous amino acids for transporter activity, could have major implications for syncytiotrophoblast metabolism and function as well as fetal development.

Targeting of metallothionein by L-homocysteine: a novel mechanism for disruption of zinc and redox homeostasis.

OBJECTIVE: L-homocysteine and/or L-homocystine interact in vivo with albumin and other extracellular proteins by forming mixed-disulfide conjugates. Because of its extremely rich cysteine content, we hypothesized that metallothionein, a ubiquitous intracellular zinc-chaperone and superoxide anion radical scavenger, reacts with L-homocysteine and that homocysteinylated-metallothionein suffers loss of function. METHODS AND RESULTS: 35S-homocysteinylated-metallothionein was resolved in lysates of cultured human aortic endothelial cells in the absence and presence of reduced glutathione by SDS-PAGE and identified by Western blotting and phosphorimaging. Using zinc-Sepharose chromatography, L-homocysteine was shown to impair the zinc-binding capacity of metallothionein even in the presence of reduced glutathione. L-Homocysteine induced a dose-dependent increase in intracellular free zinc in zinquin-loaded human aortic endothelial cells within 30 minutes, followed by the appearance of early growth response protein-1 within 60 minutes. In addition, intracellular reactive oxygen species dramatically increased 6 hours after L-homocysteine treatment. In vitro studies demonstrated that L-homocysteine is a potent inhibitor of the superoxide anion radical scavenging ability of metallothionein. CONCLUSIONS: These studies provide the first evidence that L-homocysteine targets intracellular metallothionein by forming a mixed-disulfide conjugate and that loss of function occurs after homocysteinylation. The data support a novel mechanism for disruption of zinc and redox homeostasis.

Molecular targeting by homocysteine: a mechanism for vascular pathogenesis.

Hyperhomocysteinemia is an independent risk factor for cardiovascular disease. Although there is a growing body of evidence that homocysteine plays a causal role in atherogenesis, specific mechanisms to explain the underlying pathology have remained elusive. This review focuses on chemistry unique to the homocysteine molecule to explain its inherent cytotoxicity. Thus, the high pKa of the sulfhydryl group (pKa=10.0) of homocysteine underlies its ability to form stable disulfide bonds with protein cysteine residues, and in the process, alters or impairs the function of the protein. Albumin, fibronectin, transthyretin, annexin II, and factor V have now been identified as molecular targets for homocysteine, and in the case of albumin, the mechanism of targeting has been elucidated.

Synthesis of highly fluorescent y-enyne dendrimers with four and six arms.

A first generation of dendrimeric Y-enynes with extended flexible chains was synthesized using Sonogashira coupling. Dendrimers 9 and 10 are highly fluorescent in the solid state and in solution.