Identification of a by-product of nitric oxide synthase activity in human acute brain injury with in vivo proton magnetic resonance spectroscopy

BACKGROUND AND PURPOSE: Laboratory studies have been used to identify nitric oxide as a notable mediator in neuronal death after acute brain injury. To our knowledge, this has not previously been confirmed with in vivo study in humans. Our purpose was to seek in vivo evidence for the induction of nitric oxide synthase (NOS) in human acute brain injury by using proton MR spectroscopy.

METHODS: In vitro proton MR spectra were obtained in neural extracts from 30 human cadavers, and in vivo spectra were obtained in 20 patients with acute brain injury and in a similar number of control subjects.

RESULTS: We identified a unique peak at 3.15 ppm by using in vivo proton MR spectroscopy in eight of 20 patients with acute brain injury but not in 20 healthy volunteers (P < .002). On the basis of in vitro data, we have tentatively assigned this peak to citrulline, a NOS by-product.

CONCLUSION: To our knowledge, our findings suggest, for the first time, that excitotoxicity may occur in human acute brain injury. Confirmation with the acquisition of spectra in very early acute cerebral injury would provide a rationale for the use of neuroprotective agents in these conditions, as well as a new noninvasive method for quantification.

Nitric oxide control of lower vertebrate blood vessels by vasomotor nerves

In mammals, much is understood about the endothelial and neural NO control mechanisms in the vasculature. In contrast, NO control of blood vessels in lower vertebrates is poorly understood, with the majority of research focusing on the presence of an endothelial NO system; however, its presence remains controversial. This study examined the mechanisms by which NO regulates the large blood vessels of non-mammalian vertebrates. In all species examined, the arteries and veins contained a plexus of NOS-positive perivascular nerves that included nerve bundles and fine, varicose nerve terminals. However, in the large arteries and veins of various species of fishes and amphibians, no anatomical evidence was found for endothelial NOS using both NADPH-diaphorase and eNOS immunohistochemistry. In contrast, perinuclear NOS staining was readily apparent in blue-tongue lizard, pigeon and rat, which suggested that eNOS first appeared in reptiles. Physiological analysis of NO signalling in the vascular smooth muscle of short-finned eel and cane toad could not find any evidence for endothelial NO signalling. In contrast, it appears that activation of the nitrergic vasomotor nerves is responsible for NO control of the blood vessels.

Neurally-derived nitric oxide regulates vascular tone in pulmonary and cutaneous arteries of the toad, Bufo marinus

In this study, the role of nitric oxide (NO) in regulation of the pulmocutaneous vasculature of the toad, Bufo marinus was investigated. In vitro myography demonstrated the presence of a neural NO signaling mechanism in both arteries. Vasodilation induced by nicotine was inhibited by the soluble guanylyl cyclase (GC) inhibitor, 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one, and the NO synthase (NOS) inhibitor, N^ω-nitro-_L-arginine (_L-NNA). Removal of the endothelium had no significant effect on the vasodilation. Furthermore, pretreatment with N⁵-(1-imino-3-butenyl)-_L-ornithine (vinyl-_L-NIO), a more specific inhibitor of neural NOS, caused a significant decrease in the nicotine-induced dilation. In the pulmonary artery only, a combination of _L-NNA and the calcitonin gene-related peptide (CGRP) receptor antagonist, CGRP(_8-37), completely blocked the nicotine-induced dilation. In both arteries, the vasodilation was also significantly decreased by glibenclamide, an ATP-sensitive K⁺ (K⁺_ATP) channel inhibitor. Levcromakalim, a K⁺_ATP channel opener, caused a dilation that was blocked by glibenclamide in both arteries. In the pulmonary artery, NO donor-mediated dilation was significantly decreased by pretreatment with glibenclamide. The physiological data were supported by NADPH-diaphorase histochemistry and immunohistochemistry, which demonstrated NOS in perivascular nerve fibers but not the endothelium of the arteries. These results indicate that the pulmonary and cutaneous arteries of B. marinus are regulated by NO from nitrergic nerves rather than NO released from the endothelium. The nitrergic vasodilation in the arteries appears to be caused, in part, via activation of K⁺_ATP channels. Thus, NO could play an important role in determining pulmocutaneous blood flow and the magnitude of cardiac shunting.

Mechanisms of nitric oxide-mediated neurogenic,vasodilation in mesenteric resistance arteries of toad, Bufo marinus

This study determined the role of nitric oxide (NO) in neurogenic vasodilation in mesenteric resistance arteries of the toad Bufo marinus. NO synthase (NOS) was anatomically demonstrated in perivascular nerves, but not in the endothelium. ACh and nicotine caused TTX-sensitive neurogenic vasodilation of mesenteric arteries. The ACh-induced vasodilation was endothelium-independent and was mediated by the NO/soluble guanylyl cyclase signaling pathway, inasmuch as the vasodilation was blocked by the soluble guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one and the NOS inhibitors N^ω- nitro-L-arginine methyl ester and N^ω-nitro-L-arginine. Furthermore, the ACh-induced vasodilation was significantly decreased by the more selective neural NOS inhibitor N⁵-(1-imino-3-butenyl)-L-ornithine. The nicotine-induced vasodilation was endothelium-independent and mediated by NO and calcitonin gene-related peptide (CGRP), inasmuch as pretreatment of mesenteric arteries with a combination of N^ω-nitro-L-arginine and the CGRP receptor antagonist CGRP-(8–37) blocked the vasodilation. Clotrimazole significantly decreased the ACh-induced response, providing evidence that a component of the NO vasodilation involved Ca^2+-activated K⁺ or voltage-gated K⁺ channels. These data show that NO control of mesenteric resistance arteries of toad is provided by nitrergic nerves, rather than the endothelium, and implicate NO as a potentially important regulator of gut blood flow and peripheral blood pressure.

The evolution of nitric oxide signalling in vertebrate blood vessels

Nitric oxide is one of the most important signalling molecules involved in the regulation of physiological function. It first came to prominence when it was discovered that the vascular endothelium of mammals synthesises and releases nitric oxide (NO) to mediate a potent vasodilation. Subsequently, it was shown that NO is synthesised in the endothelium by a specific isoform of nitric oxide synthase (NOS) called NOS3. Following this discovery, it was assumed that an endothelial NO/NOS3 system would be present in all vertebrate blood vessels. This review will discuss the latest genomic, anatomical and physiological evidence which demonstrates that an endothelial NO/NOS3 signalling is not ubiquitous in non-mammalian vertebrates, and that there have been key evolutionary steps that have led to the endothelial NO signalling system being a regulatory system found only in reptiles, birds and mammals. Furthermore, the emerging role of nitrite as an endocrine source of NO for vascular regulation is discussed.

NOS isoform-specific regulation of basal but not exercise-induced mitochondrial biogenesis in mouse skeletal muscle

Nitric oxide is a potential regulator of mitochondrial biogenesis. Therefore, we investigated if mice deficient in endothelial nitric oxide synthase (eNOS-/-) or neuronal NOS (nNOS-/-) have attenuated activation of skeletal muscle mitochondrial biogenesis in response to exercise. eNOS-/-, nNOS-/- and C57Bl6 (CON) mice (16.3 ± 0.2 weeks old) either remained in their cages (basal) or ran on a treadmill (16 m min-1, 5 grade) for 60 min (n = 8 per group) and were killed 6 h after exercise. Other eNOS-/-, nNOS-/- and CON mice exercise trained for 9 days (60 min per day) and were killed 24 h after the last bout of exercise training. eNOS-/- mice had significantly higher nNOS protein and nNOS-/- mice had significantly higher eNOS protein in the EDL, but not the soleus. The basal mitochondrial biogenesis markers NRF1, NRF2α and mtTFA mRNA were significantly (P< 0.05) higher in the soleus and EDL of nNOS-/- mice whilst basal citrate synthase activity was higher in the soleus and basal PGC-1α mRNA higher in the EDL. Also, eNOS-/- mice had significantly higher basal citrate synthase activity in the soleus but not the EDL. Acute exercise increased (P< 0.05) PGC-1α mRNA in soleus and EDL and NRF2α mRNA in the EDL to a similar extent in all genotypes. In addition, short-term exercise training significantly increased cytochrome c protein in all genotypes (P< 0.05) in the EDL. In conclusion, eNOS and nNOS are differentially involved in the basal regulation of mitochondrial biogenesis in skeletal muscle but are not critical for exercise-induced increases in mitochondrial biogenesis in skeletal muscle.

Vasodilator mechanisms in the dorsal aorta of the giant shovelnose ray, Rhinobatus typus (Rajiformes; Rhinobatidae)

This study investigated the nature of previous termvasodilator mechanismsnext term in the dorsal aorta of the giant shovelnose ray, Rhinobatus typus. Anatomical techniques found no evidence for an endothelial nitric oxide synthase, but neural nitric oxide synthase was found to be present in the perivascular nerve fibres of the dorsal aorta and other arteries and veins using both NADPH-diaphorase staining and immunohistochemistry with a specific neural NOS antibody. Arteries and veins both contained large nNOS-positive nerve trunks from which smaller nNOS-positive bundles branched and formed a plexus in the vessel wall. Single, varicose nNOS-positive nerve fibres were present in both arteries and veins. Within the large bundles of both arteries and veins, groups of nNOS-positive cell bodies forming microganglia were observed. Double-labelling immunohistochemistry using an antibody to tyrosine hydroxylase showed that nearly all the NOS nerves were not sympathetic. Acetylcholine always caused constriction of isolated rings of the dorsal aorta and the nitric oxide donor, sodium nitroprusside, did not mediate any dilation. Addition of nicotine (3×10−4 M) to preconstricted rings caused a vasodilation that was not affected by the nitric oxide synthase inhibitor, Image -NNA (10−4 M), nor the soluble guanylyl cyclase inhibitor, ODQ (10−5 M). This nicotine-mediated vasodilation was, therefore, not due to the synthesis and release of NO. Disruption of the endothelium significantly reduced or eliminated the nicotine-mediated vasodilation. In addition, indomethacin (10−5 M), an inhibitor of cyclooxygenases, significantly increased the time period to maximal dilation and reduced, but did not completely inhibit the nicotine-mediated vasodilation. These data support the hypothesis that a prostaglandin is released from the vascular endothelium of a batoid ray, as has been described previously in other groups of fishes. The function of the nitrergic innervation of the blood vessels is not known because nitric oxide does not appear to regulate vascular tone.

Postexercise muscle cooling enhances gene expression of PGC-1α

This study aimed to investigate the influence of localized muscle cooling on postexercise vascular, metabolic, and mitochondrial-related gene expression.

Statin-induced increases in atrophy gene expression occur independently of changes in PGC1α protein and mitochondrial content

One serious side effect of statin drugs is skeletal muscle myopathy. Although the mechanism(s) responsible for statin myopathy remains to be fully determined, an increase in muscle atrophy gene expression and changes in mitochondrial content and/or function have been proposed to play a role. In this study, we examined the relationship between statin-induced expression of muscle atrophy genes, regulators of mitochondrial biogenesis, and markers of mitochondrial content in slow- (ST) and fast-twitch (FT) rat skeletal muscles. Male Sprague Dawley rats were treated with simvastatin (60 or 80 mg·kg(-1)·day(-1)) or vehicle control via oral gavage for 14 days. In the absence of overt muscle damage, simvastatin treatment induced an increase in atrogin-1, MuRF1 and myostatin mRNA expression; however, these were not associated with changes in peroxisome proliferator gamma co-activator 1 alpha (PGC-1α) protein or markers of mitochondrial content. Simvastatin did, however, increase neuronal nitric oxide synthase (nNOS), endothelial NOS (eNOS) and AMPK α-subunit protein expression, and tended to increase total NOS activity, in FT but not ST muscles. Furthermore, simvastatin induced a decrease in β-hydroxyacyl CoA dehydrogenase (β-HAD) activity only in FT muscles. These findings suggest that the statin-induced activation of muscle atrophy genes occurs independent of changes in PGC-1α protein and mitochondrial content. Moreover, muscle-specific increases in NOS expression and possibly NO production, and decreases in fatty acid oxidation, could contribute to the previously reported development of overt statin-induced muscle damage in FT muscles.

Potential role of nitric oxide in contraction-stimulated glucose uptake and mitochondrial biogenesis in skeletal muscle

• 1. The present review discusses the potential role of nitric oxide (NO) in the: (i) regulation of skeletal muscle glucose uptake during exercise; and (ii) activation of mitochondrial biogenesis after exercise.
• 2. We have shown in humans that local infusion of an NO synthase inhibitor during exercise attenuates increases in skeletal muscle glucose uptake without affecting blood flow. Recent studies from our laboratory in rodents support these findings in humans, although rodent studies from other laboratories have yielded conflicting results.
• 3. There is clear evidence that NO increases mitochondrial biogenesis in non-contracting cells and that NO influences basal skeletal muscle mitochondrial biogenesis. However, there have been few studies examining the potential role of NO in the activation of mitochondrial biogenesis following an acute bout of exercise or in response to exercise training. Early indications are that NO is not involved in regulating the increase in mitochondrial biogenesis that occurs in response to exercise.
• 4. Exercise is considered the best prevention and treatment option for diabetes, but unfortunately many people with diabetes do not or cannot exercise regularly. Alternative therapies are therefore critical to effectively manage diabetes. If skeletal muscle NO is found to play an important role in regulating glucose uptake and/or mitochondrial biogenesis, pharmaceutical agents designed to mimic these effects of exercise may improve glycaemic control.

Mechanism of nitric oxide regulation of vascular tone in the mesenteric arteries of the cane toad, Bufo marinus

Nitric oxide control of large systemic blood vessels of the cane toad, Bufo marinus is provided by nitrergic nerves. However, the involvement of nitrergic nerves in the regulation of small blood vessels has yet to be determined. This study investigated the nitric oxide (NO) control of the mesenteric arteries (MA) of B. marinus. Immunohistochemistry and NADPH-diaphorase histochemistry demonstrated a dense plexus of nitrergic nerves in the MA of B. marinus. MAs (~ 500–700µm in diameter) were mounted in a myograph and placed under an initial tension equivalent to their normal diameter. MAs were pre-constricted with the thromboxane A2 mimetic, U46619, prior to the addition of putative, vasodilatory chemicals. Acetylcholine caused a vasodilation that was endothelium-independent, because removal of the endothelium had no effect on the dilation. The response to acetylcholine was blocked by the NOS inhibitor, L-NNA, demonstrating that the effect was NO-dependent. Interestingly, nicotine also caused a dilation that was not affected by removal of the endothelium, but was significantly inhibited by L-NNA and the calcitonin gene-related peptide (CGRP) receptor antagonist, CGRP(8–37). These findings indicate that the MA of B. marinus are controlled by NO released from nitrergic nerves. In addition, a component of the response to applied nicotine appears to be mediated CGRP, which is probably released from sensory nerves.