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. Kin and Vi 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).
Table 1.
The unidirectional transfer constants (Kin) and initial volume of distribution (Vi) determined by multiple-time uptake analysis of [14C]-sucrose into specific CNS regions.
Table 2.
The Kin values for [3H]-arginine and [3H]-ADMA values determined by single-time point analysis at a 10-minute perfusion.
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).