One of the potential causes of hypertension is endothelial dysfunction associated with a decreased production of the vasodilator nitric oxide (NO). Possible factors which may contribute to the reduced NO production include increased reactive oxygen species (eg. superoxides); increased concentrations of homocysteine; or decreased concentrations of L-arginine (cationic amino acids). L-arginine, the precursor of NO, not only increases the bioavailability of NO by increasing its production; but also by reducing the inactivation of NO by superoxides. In patients with hypertension, although fasting plasma L-arginine concentrations are elevated, L-arginine supplementation has been shown to decrease blood pressure. A possible explanation for these data may be that L-arginine uptake into cells is impaired and therefore would not be available for NO production. Indeed, studies have shown that cellular uptake of L-arginine is reduced in lymphocytes from patients with hypertension and individuals genetically predisposed to developing hypertension. However, elucidating the kinetics of L-arginine uptake into endothelial cells is fundamental to determine whether L-arginine uptake is indeed impaired.
Previous studies have shown that the uptake of cationic amino acids into endothelial cells is mediated by the high affinity/low rate y+L transporter and the low affinity/high rate y+ transporter. However, data on the kinetics, the relative contribution and physiological importance of the individual transporters in cells expressing more than one transporter, are inconsistent; as most studies determining the uptake of radiolabelled amino acids have assumed Michaelis-Menten kinetics and have calculated constants from Lineweaver-Burk reciprocal plots and Eadie-Hofstee plots. Another approach was therefore required to overcome the limitations and assumptions made in these studies. My first aim was therefore to determine the kinetics of L-arginine uptake into endothelial cells using a general non-linear approach, which allows initial rates of uptake by more than one transporter to be determined and importantly includes the actual concentrations of both the trace radiolabelled and unlabelled amino acid in the model. Furthermore, using this approach no assumptions are made regarding the type of inhibition and the concentrations of inhibitors (or activators) could be included in the model. As the model was additive, the theoretical contribution of uptake by each transporter could be modelled.
The present study used raw, rather than transformed data, in non-linear regression analysis to characterize the kinetics of L-arginine uptake into cells. I modelled the initial high affinity/low capacity and low affinity/high capacity uptake of trace L-[3H]arginine by two transporters into ECV304 and umbilical cord vein endothelial cells in the presence of a range of unlabelled L-arginine and modifiers using GraphPad Prism. The contribution of uptake by individual transporters was modelled and showed that leucine inhibited the individual transporters differently and that the inhibition was not necessarily competitive. N-ethylmaleimide inhibited only y+ transport and 2-amino-bicyclo-[2,2,1]-heptane-2-carboxylic acid may be a potential inhibitor of y+L transport. Only the absence of sodium reduced L-arginine uptake by y+L transport and reduced the Km’, whereas reducing sodium decreased L-arginine uptake by y+ transport without affecting the Km’. This non-linear modelling approach allows more than one transporter to be modelled, overcomes many of the assumptions made in reported studies and by using raw, rather than transformed data, avoids the errors inherent in methods deriving constants from the linearization of the uptake processes following Michaelian kinetics. The results of this study therefore provide explanations for discrepancies in the literature and suggest that this modelling approach better characterises the kinetics of amino acid uptake into cells.
Having elucidated the kinetics of L-arginine uptake into endothelial cells, I was then equipped to explore possible factors which could impair L-arginine uptake in hypertension. In this regard, although increases in total plasma homocysteine were thought to play a role in hypertension; large prospective clinical trials to reduce total plasma homocysteine by vitaminB6/12/folate supplementation, have failed to show beneficial effects on vascular outcomes. The effects of homocysteine on the vasculature were attributed to the reactive free sulphydryl group; however only a fraction (1.5 – 4%) of total plasma homocysteine is actually present as the free reduced sulphydryl (-SH or thiol) form. In comparison, free oxidized homocysteine, present as the disulphide, homocystine and the mixed disulphide (with cysteine) accounts for 20 – 30% of total plasma homocysteine. In the absence of a clear mechanism by which homocysteine causes vascular disease, one of the other species making up the total homocysteine may be contributing to vascular disease through a different mechanism which may not involve the free sulphydryl group.
Earlier studies demonstrated (in isolated nephrons) that the homocysteine disulphide, homocystine, shared the same membrane transporter as L-arginine (the precursor of NO), and competed for uptake with L-arginine. These studies may suggest that increased homocystine concentrations, by inhibiting L-arginine transport, and hence reducing intracellular L-arginine concentrations, may impact on NO production in other cell types. Therefore, the second aim of my study was to determine the effects of homocystine on cellular L-arginine uptake and hence on NO production.
The uptake of labelled L-[3H]arginine was measured in confluent, L-arginine depleted HUVEC and ECV304 cells with unlabelled L-arginine, without or with homocystine and modifiers. The kinetic constants were determined in Graphpad Prism using a described non-linear model of uptake for two transporters acting simultaneously. The NO specific fluorescent DAF-2 dye was used to detect NO production by the cells. Elevated physiological concentrations of 2.5μM homocystine significantly inhibited L-arginine uptake by 90% by y+L transport in both HUVEC (p<0.0005) and in ECV304 cells (p<0.05). Homocystine reduced the Kma of y+L transport in HUVEC (<0.0001) affecting uptake in a competitive-like manner. Pre-incubation of the ECV304 cells with L-arginine was able to reverse this inhibition by homocystine. In contrast, homocystine increased uptake by y+ transport in HUVEC (p<0.01). Under the experimental conditions used, effects of homocystine on the rate of NO production could not be shown. By demonstrating that homocystine nearly abolishes L-arginine uptake by y+L transport in both HUVEC and ECV304 cells, these data provide a mechanism as to how homocystine may affect L-arginine concentrations. These data would support studies to determine the association between homocystine concentrations and cardiovascular disease.
Lastly, although angiotensin-converting enzyme inhibitors (ACEI’s, as well as angiotensin II receptor antagonists) but not other classes of antihypertensive agents, have been shown to decrease oxidative stress and increase NO availability independent of blood pressure lowering effects, the mechanism is not clear. The ability of ACEI’s to decrease oxidative stress and enhance NO production has been attributed in part to the sulfhydryl groups present in some, but not all, ACEI’s. Hence the mechanisms of the effects of ACEI’s on NO production warrant further investigation, as it is possible that L-arginine transporters may play a role by enhancing L-arginine uptake into cells, and thereby increasing NO production.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:wits/oai:wiredspace.wits.ac.za:10539/12550 |
| Date | 19 March 2013 |
| Creators | Nel, Margaretha Johanna |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf, application/pdf |
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