Novel Insights Into The Transport of Cardioactive Arginine Derivatives by CAT1 (SLC7A1) and b0,+AT-rBAT (SLC7A9-SLC3A1)

Background and objectives The pathophysiological relevance of L-arginine and its derivatives, L-homoarginine and asymmetric dimethylarginine (ADMA), is an emerging focus in cardiovascular and renal diseases. Despite the growing importance of L-homoarginine as a protective cardiovascular biomarker and ADMA as a cardiovascular risk marker, understanding their metabolism and transport at the cellular and molecular levels remains a significant challenge. In particular, knowledge of the transport proteins involved in their cellular uptake and release remains patchy. Furthermore, although some key transport proteins mediating these processes have been identified, the effect of clinically used drugs on these processes remains elusive. This thesis addressed the following objectives through three complementary studies: (1) Identify transport proteins that recognize L-arginine and its derivatives as substrates and investigate their clinical associations with cardiovascular disorders. (2) Investigate the effects of pharmacological inhibition on the transport of L-arginine, L-homoarginine, and asymmetric dimethylarginine (ADMA) mediated by the cationic amino acid transporter 1 (CAT1). (3) Establishment and validation of a stably transfected human b0,+AT-rBAT-expressing cell line for uptake studies, serving as a model for tubular reabsorption. Methods In a first step, a systematic literature search was conducted to identify the transport proteins involved in the cellular uptake and release of L-arginine and its cardioactive derivatives to determine whether transporter-mediated cellular uptake and efflux of L-arginine and cardioactive arginine derivatives are critical for cardiovascular health, and to identify the transport kinetics of individual transporters affecting L-arginine and its derivatives. In a second project, the effects of 113 clinically used drugs on CAT1 (SLC7A1)-mediated transport were evaluated using cellular uptake assays for L-arginine, L-homoarginine, and ADMA. The assays were performed in a cationic amino acid transporter 1 (CAT1)-overexpressing human embryonic kidney 293 (HEK293) cell line and a control cell line (HEK293-VC). The substrates were radiolabelled and incubated in the presence and absence of the drugs. The cellular accumulation of the substrates was detected by scintillation counting and compared in both cell lines to calculate the CAT1-attributable transport. The third project involved the establishment of a stable transfection of Madin-Darby canine kidney (MDCKII) cells for the co-expression of b0,+AT and rBAT. After characterisation of this double-transfected MDCKII-b0,+AT-rBAT cell line, L-arginine, L-homoarginine and ADMA uptake were investigated in control (MDCKII-VC), single-transfected (MDCKII-b0,+AT) and double-transfected (MDCKII-b0,+AT-rBAT) cell lines. The radiolabelled compounds were administered apically. The intracellular distribution of the applied substance was compared between MDCKII-VC, MDCKII-b0,+AT and b0,+AT-rBAT cells. Results and Observations In the first part of the review, eighteen members of the solute carrier (SLC) and ATP-binding cassette (ABC) superfamily of transporters were identified as being responsible for the cellular uptake of L-arginine, L-homoarginine and ADMA. The review highlights two distinct functions by which L-arginine and its derivative transporters are associated with cardiovascular disease (CVD). Firstly, by providing L-arginine for nitric oxide (NO) production, a function predominantly performed by CAT1. Drugs may interfere with the transport of L-arginine and its derivatives, potentially contributing to adverse and beneficial effects in CVD, highlighting the need to identify potential inhibitors. Secondly, cellular transport processes help to regulate the homeostasis and disposition of L-arginine and its derivatives, for example in the kidneys which remains only incompletely understood, so far. The second part of the study showed that 23 out of 113 drugs exhibited significant (p < 0.01) inhibition or stimulation of CAT1-mediated uptake of L-arginine, L-homoarginine, and ADMA. However, the inhibitory effects were weak to moderate. These results suggest that CAT1 is subject to a strict regulatory mechanism due to the crucial role of its substrate, L-arginine, as the sole precursor for nitric oxide (NO) synthesis. No potent inhibitor of CAT1 has been identified, however, the observed verapamil-inhibited CAT1-mediated transport may be of additional interest for its potential clinical consequences. The third manuscript describes the cloning, establishment and first characterisation of MDCKII cell lines overexpressing SLC7A9-SLC3A1 (b0,+AT-rBAT). SLC3A1 and SLC7A9 mRNA expression in MDCKII cells was verified by quantitative polymerase chain reaction (PCR), while immunofluorescence microscopy showed that b0,+AT-rBAT is predominantly located in the apical membrane. Using the established cell lines, we investigated the transport properties of L-arginine and cardioactive arginine derivatives. Radiolabelled L-arginine, L-homoarginine, and ADMA uptake were higher in double-transfected MDCKII-b0,+AT cells than in single-transfected and control cells, indicating their functional status. Concentration-dependent studies showed Km values of 512.6 ± 109.3 µM for L-arginine, 197 ± 31 µM for L-homoarginine, and non-saturated transport for ADMA. L-arginine inhibits the uptake of L-homoarginine with an IC50 value of 115.8 μM. In contrast, L-arginine showed a partial inhibition of ADMA uptake. The b0,+AT-rBAT substrate, L-cystine, showed moderate inhibition of L-arginine, L-homoarginine, and ADMA uptake at physiological plasma concentrations. These results are the first to demonstrate: (i) saturable transport of L-homoarginine and significant cellular uptake of ADMA by b0,+AT-rB and (ii) differential inhibition of L-homoarginine and ADMA by L-arginine via the b0,+AT-rBAT. Conclusion This study provides new insights into the fundamental function of amino acid transporters in the patho(physiological) effects of L-arginine and its derivatives. The elucidation of the transport mechanisms of b0,+AT-rBAT brings a deeper understanding of how selective reabsorption and excretion processes regulate these essential compounds, with significant implications for therapeutic strategies in the management of cardiovascular and renal disease. In addition, the study suggests that modulation of these transport systems may lead to novel interventions to improve vascular health and manage conditions associated with elevated ADMA and reduced L-homoarginine levels. The results regarding b0,+AT-rBAT suggest that the complex functions as a transporter with high affinity and capacity for L-homoarginine and may also be able to transport ADMA. In addition, the protein b0,+AT-rBAT can discriminate L-homoarginine from ADMA and is thought to be responsible for the specific processing of L-homoarginine, which has cardioprotective properties, and ADMA, which is a toxin associated with uremia. Our results highlight the importance of the b0,+AT-rBAT transporter in modulating the bioavailability of arginine derivatives, which may have broad effects on cardiovascular and renal health. Independent achievement This research presents a novel approach to the transport of arginine derivatives by establishing MDCKII cells overexpressing human b0,+AT-rBAT. The results identify L-homoarginine and ADMA as novel substrates for b0,+AT-rBAT and contribute to our understanding of the renal transport of arginine derivatives. This study provides a fundamental basis for understanding the differential renal handling of these amino acids, which is likely to contribute to the variations in plasma and urinary concentrations observed in different clinical conditions. Furthermore, this work has implications for drug interactions, disease mechanisms, and precision medicine strategies to improve cardiovascular function by modulating arginine derivative transport.