L-Arginine and asymmetric dimethylarginine (ADMA) transport across the mouse blood-brain and blood-CSF barriers: Evidence of saturable transport at both interfaces and CNS to blood efflux

Abstract

L-Arginine is the physiological substrate for the nitric oxide synthase (NOS) family, which synthesises nitric oxide (NO) in endothelial and neuronal cells. NO synthesis can be inhibited by endogenous asymmetric dimethylarginine (ADMA). NO has explicit roles in cellular signalling and vasodilation. Impaired NO bioavailability represents the central feature of endothelial dysfunction associated with vascular diseases. Interestingly, dietary supplementation with L-arginine has been shown to alleviate endothelial dysfunctions caused by impaired NO synthesis. In this study the transport kinetics of [3H]-arginine and [3H]-ADMA into the central nervous system (CNS) were investigated using physicochemical assessment and the in situ brain/choroid plexus perfusion technique in anesthetized mice. Results indicated that L-arginine and ADMA are tripolar cationic amino acids and have a gross charge at pH 7.4 of 0.981. L-Arginine (0.00149±0.00016) has a lower lipophilicity than ADMA (0.00226±0.00006) as measured using octanol-saline partition coefficients. The in situ perfusion studies revealed that [3H]-arginine and [3H]-ADMA can cross the blood-brain barrier (BBB) and the blood-CSF barrier. [3H]-Arginine (11.6nM) and [3H]-ADMA (62.5nM) having unidirectional transfer constants (Kin) into the frontal cortex of 5.84±0.86 and 2.49±0.35 μl.min-1.g-1, respectively, and into the CSF of 1.08±0.24 and 2.70±0.90 μl.min-1.g-1, respectively. In addition, multiple-time uptake studies revealed the presence of CNS-to-blood efflux of ADMA. Self- and cross-inhibition studies indicated the presence of transporters at the BBB and the blood-CSF barriers for both amino acids, which were shared to some degree. Importantly, these results are the first to demonstrate: (i) saturable transport of [3H]-ADMA at the blood-CSF barrier (choroid plexus) and (ii) a significant CNS to blood efflux of [3H]-ADMA. Our results suggest that the arginine paradox, in other words the clinical observation that NO-deficient patients respond well to oral supplementation with L-arginine even though the plasma concentration is sufficient to saturate endothelial NOS, could be related to altered ADMA transport (efflux).

Fig 1. The percentage distribution and chemical structures of the two L-arginine microspecies and the two ADMA microspecies found at physiological pH.

Microspecies A is the major microspecies. Microspecies B is the minor microspecies.

Fig 2. The brain distribution of [³H]-arginine and [¹⁴C]-sucrose as a function of time measured using the in situ brain perfusion technique in anaesthetised mice.

Uptake is expressed as the percentage ratio of tissue to plasma (mL.100 g^-1). Each point represents the mean ± SEM of 4–7 animals. K_in and V_i values were determined as the slope and ordinate intercept of the computed regression lines where appropriate and reported in Table 1. Asterisks represent one-tailed, paired Student’s t-tests comparing mean±SEM at each time point, *p < 0.05, **p < 0.01, ***p < 0.001 (GraphPad Prism 6.0 for Mac).

Fig 3. Distribution of [³H]-arginine and [¹⁴C]-sucrose in capillary depletion samples as a function of time.

Uptake is expressed as the percentage ratio of tissue to plasma (mL.100 g^-1). Each point represents the mean ±SEM of 4–7 animals. (GraphPad Prism 6.0 for Mac). Asterisks represent one-tailed, paired Student’s t-tests comparing mean±SEM at each time point, *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 4. Distribution of [³H]-arginine and [¹⁴C]-sucrose in the CSF, pineal gland, choroid plexus and pituitary gland following in situ brain perfusion as a function of time.

Uptake is expressed as the percentage ratio of tissue or CSF to plasma (mL.100 g^-1). Each point represents the mean ± SEM of 4–7 animals. Asterisks represent one-tailed, paired Student’s t-tests comparing mean±SEM at each time point, *p < 0.05, **p < 0.01, ***p < 0.001 (GraphPad Prism 6.0 for Mac).

Fig 5. Brain distribution of [³H]-ADMA and [¹⁴C]-sucrose as a function of time.

Uptake is expressed as the percentage ratio of tissue to plasma (mL.100 g^-1). Each point represents the mean ± SEM of 5 animals. One-tailed, paired Student’s t-tests comparing mean±SEM at each time point, *p < 0.05, **p < 0.01 (GraphPad Prism 6.0 for Mac).

Fig 6. Distribution of [³H]-ADMA and [¹⁴C]-sucrose in capillary depletion samples as a function of time.

Uptake is expressed as the percentage ratio of tissue to plasma (mL.100 g^-1). Each point represents the mean ± SEM of 5 animals. One-tailed, paired Student’s t-tests comparing mean±SEM at each time point, *p < 0.05, **p < 0.01 (GraphPad Prism 6.0 for Mac).

Fig 7. Distribution of [³H]-ADMA and [¹⁴C]-sucrose in the CSF, pineal gland, choroid plexus and pituitary gland following in situ brain perfusion as a function of time.

Uptake is expressed as the percentage ratio of tissue or CSF to plasma (mL.100 g^-1). Each point represents the mean±SEM of 5 animals. One-tailed, paired Student’s t-tests comparing mean±SEM at each time point, *p< 0.05, ** p< 0.01, *** p< 0.001 (GraphPad Prism 6.0 for Mac).

Fig 8. The effect of 100μM unlabelled L-arginine on the uptake of [³H]-arginine in the brain.

Uptake is expressed as the percentage ratio of tissue to plasma (mL.100 g^-1) and is corrected for [¹⁴C]-sucrose (vascular space). Perfusion time is 10 minutes. Each bar represents the mean ± SEM of 6–7 animals (GraphPad Prism 6.0 for Mac). One-tailed, unpaired Student’s t-tests comparing means. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 9. The effect of 100μM unlabelled ADMA on the uptake of [³H]-ADMA in the brain.

Uptake is expressed as the percentage ratio of tissue to plasma (mL.100 g^-1) and is corrected for [¹⁴C]-sucrose (vascular space). Perfusion time is 10 minutes. Each bar represents the mean ± SEM of 4–5 animals (GraphPad Prism 6.0 for Mac). Unpaired Student’s t-tests comparing means. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 10. The effect of unlabelled ADMA on the regional brain uptake of [³H]-arginine (10 minute perfusion).

Uptake is expressed as the percentage ratio of tissue to plasma (mL.100 g^-1) and is corrected for [¹⁴C]-sucrose (vascular space). Perfusion time is 10 minutes. Each bar represents the mean ± SEM of 4–7 animals. Asterisks represent one-way ANOVA with Dunnett’s post-hoc tests comparing mean±SEM to control within each region/sample, *p <0.05, **p < 0.01 (GraphPad Prism 6.0 for Mac).

Fig 11. The effect of unlabelled ADMA on the distribution of [³H]-arginine in capillary depletion samples (10 minute perfusion).

Uptake is expressed as the percentage ratio of tissue to plasma (mL.100 g^-1) and is corrected for [¹⁴C]-sucrose (vascular space). Perfusion time is 10 minutes. Each bar represents the mean ± SEM of 4–7 animals. Asterisks represent one-way ANOVA with Dunnett’s post-hoc tests comparing mean±SEM to control within each region/sample, *p < 0.05 (GraphPad Prism 6.0 for Mac).

Fig 12. The effect of unlabelled ADMA on the distribution of [³H]-arginine in CSF, choroid plexus and CVOs (10 minute perfusion).

Uptake is expressed as the percentage ratio of tissue or CSF to plasma (mL.100 g^-1). Perfusion time is 10 minutes. Each bar represents the mean ± SEM of 4–7 animals. Asterisks represent one-way ANOVA with Dunnett’s post-hoc tests comparing mean±SEM to control within each region/sample, **p < 0.01 (GraphPad Prism 6.0 for Mac).