Blocking a vicious cycle nNOS/peroxynitrite/AMPK by S-nitrosoglutathione: implication for stroke therapy

Abstract

BackgroundStroke immediately sets into motion sustained excitotoxicity and calcium dysregulation, causing aberrant activity in neuronal nitric oxide synthase (nNOS) and an imbalance in the levels of nitric oxide (NO). Drugs targeting nNOS-originated toxicity may therefore reduce stroke-induced damage. Recently, we observed that a redox-modulating agent of the NO metabolome, S-nitrosoglutathione (GSNO), confers neurovascular protection by reducing the levels of peroxynitrite, a product of aberrant NOS activity. We therefore investigated whether GSNO-mediated neuroprotection and improved neurological functions depend on blocking nNOS/peroxynitrite-associated injurious mechanisms using a rat model of cerebral ischemia reperfusion (IR).ResultsIR increased the activity of nNOS, the levels of neuronal peroxynitrite and phosphorylation at Ser1412 of nNOS. GSNO treatment of IR animals decreased IR-activated nNOS activity and neuronal peroxynitrite levels by reducing nNOS phosphorylation at Ser1412. The Ser1412 phosphorylation is associated with increased nNOS activity. Supporting the notion that nNOS activity and peroxynitrite are deleterious following IR, inhibition of nNOS by its inhibitor 7-nitroindazole or reducing peroxynitrite by its scavenger FeTPPS decreased IR injury. GSNO also decreased the activation of AMP Kinase (AMPK) and its upstream kinase LKB1, both of which were activated in IR brain. AMPK has been implicated in nNOS activation via Ser1412 phosphorylation. To determine whether AMPK activation is deleterious in the acute phase of IR, we treated animals after IR with AICAR (an AMPK activator) and compound c (an AMPK inhibitor). While AICAR potentiated, compound c reduced the IR injury.ConclusionsTaken together, these results indicate an injurious nNOS/peroxynitrite/AMPK cycle following stroke, and GSNO treatment of IR inhibits this vicious cycle, resulting in neuroprotection and improved neurological function. GSNO is a natural component of the human body, and its exogenous administration to humans is not associated with any known side effects. Currently, the FDA-approved thrombolytic therapy suffers from a lack of neuronal protective activity. Because GSNO provides neuroprotection by ameliorating stroke’s initial and causative injuries, it is a candidate of translational value for stroke therapy.

Targeting the nNOS/peroxynitrite/calpain system to confer neuroprotection and aid functional recovery in a mouse model of TBI

“…Taken together, these observations indicate a disturbed NO metabolome in TBI. This disturbed NO metabolome in TBI is supported by previous studies from our laboratory in TBI (Khan et al, 2011) and stroke (Khan et al, 2012; Khan et al, 2015b) and the current results, which show increased enzymatic nNOS activity (Fig 1B) and sustained high levels of peroxynitrite for a prolonged periods (Figs 2D–d and 4B–b). We investigated the mechanisms of the efficacy of GSNO for TBI therapy because it corrected not only the disturbed NO metabolome (Fig 1) and NO/S-nitrosylation-based cellular functions (Khan et al, 2005; Khan et al, 2011; Khan et al, 2015a; Sakakima et al, 2012) but also provided neuroprotection and improved neurological functions, as shown in Fig 5.…”

“…In this cell free system, a treatment with SIN-1, a peroxynitrite-producing agent (Khan et al, 2006; Pacher et al, 2007), significantly and dose dependently increased whereas GSNO significantly and dose dependently decreased calpain activity (Fig 3A). Reversal of the SIN-1-mediated enhanced calpain activity by FeTPPS, a peroxynitrite scavenger (Khan et al, 2015b; Thiyagarajan et al, 2004), supports the role of peroxynitrite for increasing calpain activity, likely via the mechanism of 3-nitrotyrosination (Fig 3B). Blocking the GSNO-mediated reduced calpain activity by HgCl 2 , a Cys-NO bond cleaving agent (Cook et al, 1996), supports the role of GSNO in decreasing calpain activity, likely via the mechanisms of S-nitrosylation (Fig 3B).…”

“…The dose of GSNO treatment was based on our previously reported dose response curve study in a rat model of TBI and SCI (Chou et al, 2011; Khan et al, 2009; Khan et al, 2011). The effective dose of FeTPPS was determined based on the reported dose in a stroke IR model (Khan et al, 2015b; Thiyagarajan et al, 2004). The selected dose of both GSNO and FeTPPS did not alter physiologic parameters, including BP, HR, and temperature, measured at 1 hour following GSNO or FeTPPS treatment.…”

“…In the traumatic penumbra area from the ipsilateral injured brain tissue, Western blot was performed using antibodies against calpain 1, calpain 2 (Cell signaling Technology, Danvers, MA), alpha spectrin (Santa Cruz Biotechnology, Santa Cruz, CA), 3-NT, nNOS, phospho nNOS Ser 1417 , equivalent to human Ser 1412 (Abcam Inc., Cambridge, MA) and β-actin, as described earlier (Khan et al, 2012; Khan et al, 2015b). Protein concentrations were determined using protein assay dye from Bio-Rad Laboratories (Hercules, CA).…”

AMPK in the central nervous system: physiological roles and pathological implications

“…These data support the hypothesis that AMPK activation in the acute phase of stroke is associated with injury. 108 To corroborate the idea that AMPK activation is harmful in ischemic stroke, preclinical studies demonstrated that mice deficient in AMPKα2 have less injury and show a reduced total infarct volume compared with wild-type littermates in an MCAO reperfusion model. AMPKα1-knockout mice have no difference in injury compared with wild-type mice, suggesting that AMPKα2 isoform plays a more significant role in the damaging response of AMPK activation in ischemic brain.…”