Optogenetic Stimulation Reduces Neuronal Nitric Oxide Synthase Expression After Stroke

INTRODUCTION Poststroke optogenetic stimulation has been shown to enhance neurovascular coupling and functional recovery. Neuronal nitric oxide synthase (nNOS) has been implicated as a key regulator of neurovascular response in acute stroke but its role in subacute recovery remains unclear. Here we investigate nNOS expression in stroke mice undergoing optogenetic stimulation of the contralesional lateral cerebellar nucleus (cLCN). We also examine the effects of nNOS inhibition on functional recovery using a pharmacological inhibitor targeting nNOS. METHODS Transgenic Thy1-ChR2-YFP male mice (10-12 wk) were used. Stereotaxic surgery was performed to implant a fiber cannula in the cLCN and animals underwent intraluminal middle cerebral artery suture occlusion (30 min). Optogenetic stimulation began at poststroke (PD) day 5 and continued until PD14. Sensorimotor tests were used to assess behavioral recovery at PD4, 7, 10, and 14. At PD15, primary motor cortex from both ipsi- and contralesional motor cortex (iM1, cM1) were dissected. nNOS mRNA and protein levels were examined using quantitative polymerase chain reaction and western blot. In another set of studies, nNOS inhibitor ARL 17477 dihydrochloride (10 mg/kg, intraperitoneally) was administered daily between PD5-14 and functional recovery was evaluated using sensorimotor tests. RESULTS cLCN stimulated stroke mice demonstrated significant improvement in speed (cm/s) on the rotating beam task at PD10 and 14 day (P < .05, P < .001 respectively). nNOS mRNA and protein expression was significantly and selectively decreased in cM1 of cLCN stimulated mice (P < .05). The reduced nNOS expression in cM1 was negatively correlated with improved recovery (R2 = −0.839, Pearson P = .009). nNOS inhibitor-treated stroke mice exhibited a significant functional improvement in speed at PD10, when compared to stroke mice receiving vehicle (saline) (P < .05). CONCLUSION Our results suggest that nNOS may play a maladaptive role in poststroke recovery. Optogenetic stimulation of cLCN and systemic nNOS inhibition produce functional benefits after stroke.

Neuronal nitric oxide synthase-derived hydrogen peroxide effect in grafts used in human coronary bypass surgery

Recently, H2O2 has been identified as the endothelium-dependent hyperpolarizing factor (EDHF), which mediates flow-induced dilation in human coronary arteries. Neuronal nitric oxide synthase (nNOS) is expressed in the cardiovascular system and, besides NO, generates H2O2. The role of nNOS-derived H2O2 in human vessels is so far unknown. The present study was aimed at investigating the relevance of nNOS/H2O2 signaling in the human internal mammary artery (IMA) and saphenous vein (SV), the major conduits used in coronary artery bypass grafting. In the IMA, but not in the SV, ACh (acetylcholine)-induced vasodilatation was decreased by selective nNOS inhibition with TRIM or Inhibitor 1, and by catalase, which specifically decomposes H2O2. Superoxide dismutase (SOD), which generates H2O2 from superoxide, decreased the vasodilator effect of ACh on SV. In the IMA, SOD diminished phenylephrine-induced contraction in endothelium-containing, but not in endothelium-denuded vessels. Importantly, while exogenous H2O2 produced vasodilatation in IMA, it constricted SV. ACh increased H2O2 production in both sets of vessels. In the IMA, the increase in H2O2 was inhibited by catalase and nNOS blockade. In SV, H2O2 production was abolished by catalase and reduced by nNOS inhibition. Immunofluorescence experiments showed the presence of nNOS in the vascular endothelium and smooth muscle cells of both the IMA and SV. Together, our results clearly show that H2O2 induced endothelium-dependent vascular relaxation in the IMA, whereas, in the SV, H2O2 was a vasoconstrictor. Thus, H2O2 produced in the coronary circulation may contribute to the susceptibility to accelerated atherosclerosis and progressive failure of the SV used as autogenous graft in coronary bypass surgery.

Abstract WP431: Functionally Distinct Neuronal Nitric Oxide Synthase Expressed in the Brain Microvascular Endothelial Cells Mediates Anoxic-injury to Blood-Brain Barrier

Objective: Mice with genetic deletion of endothelial (eNOS; protective) and neuronal (nNOS; detrimental) nitric oxide synthase isoforms exhibit dramatically opposite consequences of ischemic brain injury. nNOS has been identified recently in endothelial cells, however, its functional significance is unclear. Our objective was to identify nNOS and characterize its functional role in primary brain microvascular endothelial cells (MECs). Methods and Results: MECs from humans (hMECs), rats (rMECs), and mice (mMECs) along with cultured primary rat cortical neurons were used. In addition, rat brain microvessels were freshly isolated. Transendothelial electrical resistance (TEER) measurements of monolayers of hMECs cultured in transwells were used to quantitate in vitro blood-brain barrier (BBB) integrity. Immunocytochemistry identified von Willebrand factor, eNOS, and nNOS in MECs but stained negative for glial (GFAP) and neuronal (Neu1) markers. PCR studies confirmed the expression of eNOS and nNOS mRNA in MECs and microvessels. We utilized electron spin resonance spectrometry to measure reactive oxygen species (ROS) (1-Hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethyl-pyrrolidine; CMH) and NO (colloid Fe(DETC)2). Inhibition of nNOS (N-ω-Propyl-L-arginine and ARL-17477) reduced ROS but increased NO levels in MECs and rat brain microvessels. In contrast, eNOS inhibitor (L-N5-(1-Iminoethyl)ornithine) increased ROS but reduced NO levels. Inhibition of nNOS in neurons, similarly increased ROS and decreased NO levels. siRNA targeting rat nNOS in rMECs was able to knockdown nNOS mRNA as well as ROS levels. BBB studies of hMECs treated with NOS inhibitors followed by oxygen-glucose deprivation (OGD) revealed that nNOS inhibition increased TEER at baseline and promoted TEER recovery following OGD. In contrast, eNOS inhibition had no effect on TEER at baseline but weakly albeit transiently helps in the post-OGD recovery of BBB function. Conclusions: Thus, we identified a constitutively active nNOS in MECs that is functionally distinct from the nNOS isoform expressed in neurons and eNOS. In addition, nNOS inhibition enhances the BBB integrity and affords protection against anoxic-injury induced impairment of BBB function.

Bimodal effect of oxidative stress in internal anal sphincter smooth muscle

Changes in oxidative stress may affect basal tone and relaxation of the internal anal sphincter (IAS) smooth muscle in aging. We examined this issue by investigating the effects of the oxidative stress inducer 6-anilino-5,8-quinolinedione (LY-83583) in basal as well as U-46619-stimulated tone, and nonadrenergic, noncholinergic (NANC) relaxation in rat IAS. LY-83583, which works via generation of reactive oxygen species in living cells, produced a bimodal effect in IAS tone: lower concentrations (0.1 nM to 10 μM) produced a concentration-dependent increase, while higher concentrations (50–100 μM) produced a decrease in IAS tone. An increase in IAS tone by lower concentrations was associated with an increase in RhoA/Rho kinase (ROCK) activity. This was evident by the increase in RhoA/ROCK in the particulate fractions, in ROCK activity, and in the levels of phosphorylated (p) Thr696-myosin phosphatase target subunit 1 and pThr18/Ser19-20-kDa myosin light chain. Conversely, higher concentrations of LY-83583 produced inhibitory effects on RhoA/ROCK. Interestingly, both the excitatory and inhibitory effects of LY-83583 in the IAS were reversed by superoxide dismutase. The excitatory effects of LY-83583 were found to resemble those with neuronal nitric oxide synthase (nNOS) inhibition by l-NNA, since it produced a significant increase in the IAS tone and attenuated NANC relaxation. These effects of LY-83583 and l-NNA were reversible by l-arginine. This suggests the role of nNOS inhibition and RhoA/ROCK activation in the increase in IAS tone by LY-83583. These data have important implications in the pathophysiology and therapeutic targeting of rectoanal disorders, especially associated with IAS dysfunction.

Sex- and limb-specific differences in the nitric oxide-dependent cutaneous vasodilation in response to local heating

Local heating of the skin is commonly used to assess cutaneous microvasculature function. Controversy exists as to whether there are limb or sex differences in the nitric oxide (NO)-dependent contribution to this vasodilation, as well as the NO synthase (NOS) isoform mediating the responses. We tested the hypotheses that 1) NO-dependent vasodilation would be greater in the calf compared with the forearm; 2) total NO-dependent dilation would not be different between sexes within limb; and 3) women would exhibit greater neuronal NOS (nNOS)-dependent vasodilation in the calf. Two microdialysis fibers were placed in the skin of the ventral forearm and the calf of 19 (10 male and 9 female) young (23 ± 1 yr) adults for the local delivery of Ringer solution (control) or 5 mM Nω-propyl-l-arginine (NPLA; nNOS inhibition). Vasodilation was induced by local heating (42°C) at each site, after which 20 mM NG-nitro-l-arginine methyl ester (l-NAME) was perfused for within-site assessment of NO-dependent vasodilation. Cutaneous vascular conductance (CVC) was calculated as laser-Doppler flux/mean arterial pressure and normalized to maximum (28 mM sodium nitroprusside, 43°C). Total NO-dependent vasodilation in the calf was lower compared with the forearm in both sexes (Ringer: 42 ± 5 vs. 62 ± 4%; P < 0.05; NPLA: 37 ± 3 vs. 59 ± 5%; P < 0.05) and total NO-dependent vasodilation was lower in the forearm for women (Ringer: 52 ± 6 vs. 71 ± 4%; P < 0.05; NPLA: 47 ± 6 vs. 68 ± 5%; P < 0.05). NPLA did not affect total or NO-dependent vasodilation across limbs in either sex ( P > 0.05). These data suggest that the NO-dependent component of local heating-induced cutaneous vasodilation is lower in the calf compared with the forearm. Contrary to our original hypothesis, there was no contribution of nNOS to NO-dependent vasodilation in either limb during local heating.

Role of neuronal nitric oxide in the inhibition of sympathetic vasoconstriction in resting and contracting skeletal muscle of healthy rats

Isoform-specific nitric oxide (NO) synthase (NOS) contributions to NO-mediated inhibition of sympathetic vasoconstriction in resting and contracting skeletal muscle are incompletely understood. The purpose of the present study was to investigate the role of neuronal NOS (nNOS) in the inhibition of sympathetic vasoconstriction in resting and contracting skeletal muscle of healthy rats. We hypothesized that acute pharmacological inhibition of nNOS would augment sympathetic vasoconstriction in resting and contracting skeletal muscle, demonstrating that nNOS is primarily responsible for NO-mediated inhibition of sympathetic vasoconstriction. Sprague-Dawley rats ( n = 13) were anesthetized and instrumented with an indwelling brachial artery catheter, femoral artery flow probe, and lumbar sympathetic chain stimulating electrodes. Triceps surae muscles were stimulated to contract rhythmically at 60% of maximal contractile force. In series 1 ( n = 9), the percent change in femoral vascular conductance (%FVC) in response to sympathetic stimulations delivered at 2 and 5 Hz was determined at rest and during muscle contraction before and after selective nNOS blockade with S-methyl-l-thiocitrulline (SMTC, 0.6 mg/kg iv) and subsequent nonselective NOS blockade with Nω-nitro-l-arginine methyl ester (l-NAME, 5 mg/kg iv). In series 2 ( n = 4), l-NAME was injected first, and then SMTC was injected to determine if the effect of l-NAME on constrictor responses was influenced by selective nNOS inhibition. Sympathetic stimulation decreased FVC at rest (−25 ± 7 and −44 ± 8%FVC at 2 and 5 Hz, respectively) and during contraction (−7 ± 3 and −19 ± 5%FVC at 2 and 5 Hz, respectively). The decrease in FVC in response to sympathetic stimulation was greater in the presence of SMTC at rest (−32 ± 6 and −49 ± 8%FVC at 2 and 5 Hz, respectively) and during contraction (−21 ± 4 and −28 ± 4%FVC at 2 and 5 Hz, respectively). l-NAME further increased ( P < 0.05) the sympathetic vasoconstrictor response at rest (−47 ± 4 and −60 ± 6%FVC at 2 and 5 Hz, respectively) and during muscle contraction (−33 ± 3 and −40 ± 6%FVC at 2 and 5 Hz, respectively). The effect of l-NAME was not altered by the order of nNOS inhibition. These data demonstrate that NO derived from nNOS and endothelial NOS contribute to the inhibition of sympathetic vasoconstriction in resting and contracting skeletal muscle.