Maintenance of cytosolic calcium is crucial to extend l-arginine therapeutic benefits during continuous dosing

The therapeutic benefits associated with short-term l-arginine supplementation are lost during continuous dosing. AMP-activated protein kinase (AMPK) functional modulation has been correlated with l-arginine therapeutic effectiveness, and with tolerance development during continuous supplementation. However, the metabolic link that is responsible for AMPK functional modulation during continuous l-arginine exposure is currently not known. To explore this, we incubated HUVECs for 7 days with 100 μmol/L l-arginine, in the presence or absence of other agents; and monitored their effects for eNOS function, and on tolerance sparing effects (viz, cellular glucose accumulation, and oxidative stress). HUVEC co-incubation with 100 μmol/L l-arginine and ≤1200 mg/mL calcium (Ca2+) for 7 days avoided tolerance development, with an at least 1-fold increase in the eNOS and AMPK functional activity; and an 1-fold increase in overall cellular glucose uptake. The overall cellular cytosolic Ca2+was below 200 nmol/L, with no change in cellular glucose and superoxide/peroxynitrite (O2
- −/ONOO−) level from control. However, tolerance sparing effects of at least 70% decrease in eNOS and AMPK functional response, with an 1-fold reduction in glucose uptake, and at least 2-fold increase in O2
- −/ONOO− were observed in cells exposed for 7 days to 100 μmol/L l-arginine at Ca2+co-incubation concentration of >1200 mg/mL. The >1200 mg/mL Ca2+ co-incubation condition, also improved the overall cellular Ca2+to >200 nmol/L. Similar tolerance response was observed in cells co-treated with 100 μmol/L l-arginine and ≤1200 mg/mL Ca2+ in the presence of Ca2+ influx inhibitor (20 μmol/L 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetra acetic acid), or eNOS activity inhibitor (30 μmol/L l-NG-nitroarginine methyl ester). No tolerance response was seen in cells incubated for 7 days with 100 μmol/L l-arginine and ≤1200 mg/mL Ca2+; even in the presence of the inhibitor for cellular glucose induction (30 μmol/L 5-chloro-2-(n-(2,5-dichlorobenzenesulfonamide))-benzoxazole). The present study thus provides the first definitive evidence that shows the need to maintain cytosolic Ca2+ within a threshold limit of less than 200 nmol/L to extend l-arginine therapeutic efficacy during continuous dosing, without any potential tolerance development.