Belinda A. Ahlers

Impaired l-Arginine Transport and Endothelial Function in Hypertensive and Genetically Predisposed Normotensive Subjects

Background—Impaired endothelium-dependent NO-mediated vasodilation is a key feature of essential hypertension and may precede the increase in blood pressure. We investigated whether transport of the NO precursor l-arginine is related to decreased endothelial function. Methods and Results—Radiotracer kinetics ([3H]l-arginine) were used to measure forearm and peripheral blood mononuclear cell arginine uptake in hypertensive subjects (n=12) and in 2 groups of healthy volunteers with (n=15) and without (n=15) a family history of hypertension. In conjunction, forearm blood flow responses to acetylcholine and sodium nitroprusside were measured before and after a supplemental intra-arterial infusion of l-arginine. In vivo and in vitro measures of l-arginine transport were substantially reduced in the essential hypertension and positive family history groups compared with the negative family history group; however, no difference was detected in peripheral blood mononuclear cell mRNA or protein expression levels for the cationic amino acid transporter CAT-1. Plasma concentrations of l-arginine and NG,NG′-dimethylarginine (ADMA) did not differ between groups. l-Arginine supplementation improved the response to acetylcholine only in subjects with essential hypertension and positive family history. Conclusions—Similar to their hypertensive counterparts, normotensive individuals at high risk for the development of hypertension are characterized by impaired l-arginine transport, which may represent the link between a defective l-arginine/NO pathway and the onset of essential hypertension. The observed transport defect is not due to apparent alterations in CAT-1 expression or elevated endogenous ADMA.

In Vivo and In Vitro Evidence for Impaired Arginine Transport in Human Heart Failure

BackgroundThe clinical features of congestive heart failure (CHF) result from a complex interaction between reduced ventricular function, neurohormonal activation, and impaired endothelial function. Although endothelial dysfunction has been well documented, the mechanisms that contribute to this abnormality remain unknown. Recent studies, however, indicate a potential therapeutic role for supplemental l-arginine, suggesting the presence of an underlying disorder of l-arginine metabolism. Methods and ResultsWe used 2 complementary approaches to assess l-arginine transport in control subjects and patients with CHF. During a steady-state intra-arterial infusion of [3H]l-arginine (100 nCi/min), forearm clearance of [3H]l-arginine was significantly reduced in CHF patients compared with forearm kinetics in control subjects (64±2 versus 133±14 mL/min, P =0.002). In conjunction with this, [3H]l-arginine uptake by peripheral blood mononuclear cells (PBMCs) was also substantially reduced in heart failure patients compared with controls (Vmax 10.1±1.3 versus 49.8±7.1 pmol/105 cells per 5 minutes, P <0.001). In association with this finding, we observed a 76% (P <0.01) reduction in mRNA expression for the cationic amino acid transporter CAT-1, as assessed by ribonuclease protection assay. ConclusionsThese data document both in vivo and in vitro evidence for a marked depression of l-arginine transport in human CHF and therefore provide an explanation for the restorative actions of supplemental l-arginine on vascular function in CHF.

Reduced Myocardial and Systemic l-Arginine Uptake in Heart Failure

Abstract— Altered nitric oxide (NO) bioavailability has been ascribed an important role in the pathophysiology of congestive heart failure (CHF). In the peripheral vasculature, we recently demonstrated a depression of l-arginine transport in association with pharmacological evidence of reduced endothelial function. In contrast, increased myocardial NO generation has been proposed to account for a component of the reduced myocardial contractility in CHF, although this remains controversial. We determined the whole body clearance rate and cardiac fractional extraction of l-arginine during a steady-state intravenous infusion of [3H]l-arginine (300 nCi/min) in 9 healthy control subjects and 7 patients with moderate to severe CHF. In patients with CHF, there was a 30% reduction in the transcardiac extraction of [3H]l-arginine compared with controls (P <0.05), which was accompanied by a trend toward reduced [3H]l-citrulline release (P =0.06). In conjunction, the systemic clearance rate of [3H]l-arginine was significantly lower in patients with CHF (778±148 versus 1278±144 mL/min, P <0.05). In association with these biochemical indices, we observed a 38% reduction (P <0.05) in the mRNA expression of the cationic amino acid transporter CAT-1 in ventricular myocardial samples from patients with CHF compared with healthy unused donor myocardium, whereas myocardial NOS enzymatic activity and NOS protein were unchanged. These data indicate the presence of a significant reduction in the myocardial uptake of l-arginine in patients with CHF. Furthermore, this abnormality seems to be part of a systemic downregulation of l-arginine transport.

An age-related decline in endothelial function is not associated with alterations in L-arginine transport in humans.

Objectives Endothelial dysfunction is established in aged individuals; however, the mechanism(s) are not fully elucidated. We have previously identified l-arginine transport as a potential rate-limiting factor in nitric oxide (NO) production in heart-failure patients, characterized with endothelial dysfunction. We therefore aimed to investigate whether the age-related decline in endothelial function is due to reduced transport of the NO precursor, l-arginine. Methods Thirty-seven healthy males aged between 19 and 69 were recruited. Throughout 40 min of intra-arterial (i.a.) infusion of [3H]l-arginine (100 nCi/min), venous blood samples were withdrawn for the determination of l-arginine clearance. Venous occlusion plethysmography was then used to record the forearm blood flow responses to i.a. infusions of acetylcholine (ACh; 9.25 and 37 μg/min) and sodium nitroprusside (SNP; 2 and 8 μg/min). Results While ACh-induced vasodilation decreased with age (37 μg/min; young 15.87 ± 1.30, middle-aged 9.59 ± 1.33, older 10.42 ± 1.12 ml/min per 100 ml tissue; P = 0.001), there was no change in forearm [3H]l-arginine clearance (young 126.08 ± 19.05, middle-aged 122.47 ± 20.96, older 126.56 ± 19.56 ml/min; NS). Further [3H]l-arginine uptake studies in isolated peripheral blood mononuclear cells supported our in vivo findings, demonstrating no difference in [3H]l-arginine transport across the age spectrum. Conclusions The present study excludes the hypothesis of impaired l-arginine transport as a potential mechanism for the age-related decline in endothelial function.

Phospholemman regulates cardiac Na+/Ca2+ exchanger by interacting with the exchanger's proximal linker domain

Phospholemman (PLM) belongs to the FXYD family of small ion transport regulators. When phosphorylated at Ser(68), PLM inhibits cardiac Na(+)/Ca(2+) exchanger (NCX1). We previously demonstrated that the cytoplasmic tail of PLM interacts with the proximal intracellular loop (residues 218-358), but not the transmembrane (residues 1-217 and 765-938) or Ca(2+)-binding (residues 371-508) domains, of NCX1. In this study, we used intact Na(+)/Ca(2+) exchanger with various deletions in the intracellular loop to map the interaction sites with PLM. We first demonstrated by Western blotting and confocal immunofluorescence microscopy that wild-type (WT) NCX1 and its deletion mutants were expressed in transfected HEK-293 cells. Cotransfection with PLM and NCX1 (or its deletion mutants) in HEK-293 cells did not decrease expression of NCX1 (or its deletion mutants). Coexpression of PLM with WT NCX1 inhibited NCX1 current (I(NaCa)). Deletion of residues 240-679, 265-373, 250-300, or 300-373 from WT NCX1 resulted in loss of inhibition of I(NaCa) by PLM. Inhibition of I(NaCa) by PLM was preserved when residues 229-237, 270-300, 328-330, or 330-373 were deleted from the intracellular loop of NCX1. These results suggest that PLM mediated inhibition of I(NaCa) by interacting with two distinct regions (residues 238-270 and 300-328) of NCX1. Indeed, I(NaCa) measured in mutants lacking residues 238-270, 300-328, or 238-270 + 300-328 was not affected by PLM. Glutathione S-transferase pull-down assays confirmed that PLM bound to fragments corresponding to residues 218-371, 218-320, 218-270, 238-371, and 300-373, but not to fragments encompassing residues 250-300 and 371-508 of NCX1, indicating that residues 218-270 and 300-373 physically associated with PLM. Finally, acute regulation of I(NaCa) by PLM phosphorylation observed with WT NCX1 was absent in 250-300 deletion mutant but preserved in 229-237 deletion mutant. We conclude that PLM mediates its inhibition of NCX1 by interacting with residues 238-270 and 300-328.

beta-Adrenoceptor-mediated, nitric-oxide-dependent vasodilatation is abnormal in early hypertension: restoration by L-arginine.

Background It is unknown whether β-adrenoceptor-mediated vasodilatation is altered in early hypertension and whether it can be modulated by l-arginine. Methods and design We measured changes in forearm blood flow by plethysmography in response to acetylcholine (9 and 37 μg/min), sodium nitroprusside (200 and 800 ng/min) and the β-receptor agonist, isoproterenol (50 and 200 ng/min) in 12 patients with essential hypertension (group EH) and in healthy volunteers with (group PFH; n = 14) and without (group NFH; n = 14) a family history of essential hypertension, before and during concomitant infusion of l-arginine (10 μmol/min). In five individuals from each group, infusion of acetylcholine and isoproterenol was repeated during the concurrent blockade of nitric oxide synthesis by NG-monomethyl-l-arginine (l-NMMA; 4 μmol/min). Results The response to acetylcholine was reduced in groups EH and PFH compared with group NFH (both P < 0.05), whereas the vasodilator effects of isoproterenol and sodium nitroprusside were similar in all three groups. Acetylcholine- and isoproterenol-induced vasodilatation did not change during infusion of the nitric oxide precursor, l-arginine, in group NFH, but were significantly enhanced by l-arginine in groups PFH and EH [forearm blood flow before and after isoproterenol 200 ng/min: group PFH 11.8 ± 1.02 and 13.3 ± 1.08 ml/min, respectively (P < 0.05); group EH: 11.3 ± 1.57 and 14.9 ± 1.91 ml/min, respectively (P < 0.01)]. Co-infusion of l-NMMA blunted the response to acetylcholine and isoproterenol in group NFH (P < 0.05), but did not significantly modify vasodilatation in groups PFH and EH. Conclusions Although β-adrenergic vasodilatation seemed to be unaltered in early hypertension, l-arginine enhanced the response to isoproterenol, whereas concomitant inhibition of nitric oxide synthase by l-NMMA had no significant effect. These findings suggest that the nitric oxide component of isoproterenol-mediated vasodilatation is impaired in early hypertension and possibly compensated by increased β-adrenoceptor responsiveness of smooth muscle cells. In this setting, supplementation of the nitric oxide precursor, l-arginine, enhances the vasodilator response to β-adrenergic stimulation.

l-Arginine Transport in Humans With Cortisol-Induced Hypertension

Abstract— A deficient l‐arginine–nitric oxide system is implicated in cortisol‐induced hypertension. We investigate whether abnormalities in l‐arginine uptake contribute to this deficiency. Eight healthy men were recruited. Hydrocortisone acetate (50 mg) was given orally every 6 hours for 24 hours after a 5‐day fixed‐salt diet (150 mmol/d). Crossover studies were performed 2 weeks apart. Thirty milliliters of blood was obtained for isolation of peripheral blood mononuclear cells after each treatment period. l‐Arginine uptake was assessed in mononuclear cells incubated with l‐arginine (1 to 300 &mgr;mol/L), incorporating 100 nmol/L [3H]‐l‐arginine for a period of 5 minutes at 37°C. Forearm [3H]‐l‐arginine extraction was calculated after infusion of [3H]‐l‐arginine into the brachial artery at a rate of 100 nCi/min for 80 minutes. Deep forearm venous samples were collected for determination of l‐arginine extraction. Plasma cortisol concentrations were significantly raised during the active phase (323±43 to 1082±245 mmol/L, P <0.05). Systolic blood pressure was elevated by an average of 7 mm Hg. Neither l‐arginine transport into mononuclear cells (placebo vs active, 26.3±3.6 vs 29.0±2.1 pmol/10 000 cells per 5 minutes, respectively, at an l‐arginine concentration of 300 &mgr;mol/L) nor l‐arginine extraction in the forearm (at 80 minutes, placebo vs active, 1 868 904±434 962 vs 2 013 910±770 619 disintegrations per minute) was affected by cortisol treatment; ie, that l‐arginine uptake is not affected by short‐term cortisol treatment. We conclude that cortisol‐induced increases in blood pressure are not associated with abnormalities in the l‐arginine transport system.

Protein kinase C mediated inhibition of endothelial L-arginine transport is mediated by MARCKS protein.

The endothelium plays a vital role in the maintenance of vascular tone and structural vascular integrity, principally mediated via the actions of nitric oxide (NO). L-arginine is the immediate substrate for NO synthesis, and the availability of extracellular L-arginine is critical for the production of NO. Activation of protein kinase C (PKC) dependent signalling pathways are a feature of a number of cardiovascular disease states, and in this study we aimed to systematically evaluate the mechanism by which PKC regulates L-arginine transport in endothelial cells. In response to PKC activation (PMA 100 nM, 30 min), [(3)H]L-arginine uptake by bovine aortic endothelial cells (BAEC) was reduced to 45+4% of control (p<0.05). This resulted from a 53% reduction in the Vmax (p<0.05), with no change in the K(m) for L-arginine. Western blot analysis and confocal microscopy revealed no change in the expression or membrane distribution of CAT-1, the principal BAEC L-arginine transporter. Moreover in (32)P-labeling studies, PMA exposure did not result in CAT-1 phosphorylation. We therefore explored the possibility that PKC altered and interaction with MARCKS protein, a candidate membrane associated protein. By co-immunoprecipitation we show that CAT-1 interacts with, a membrane associated protein, that was significantly inhibited by PKC activation (p<0.05). Moreover antisense inhibition of MARCKS abolished the PMA effect on L-arginine transport. PKC dependent mechanisms regulate the transport of L-arginine, mediated via process involving MARCKS.